Detection of Early-Stage Pancreatic Adenocarcinoma

ABSTRACT

Described herein are compositions and methods of use of anti-pancreatic cancer antibodies or fragments thereof, such as murine, chimeric, humanized or human PAM4 antibodies. The subject antibodies show a number of novel and useful diagnostic characteristics, such as binding with high specificity to pancreatic and other cancers, but not to normal pancreatic tissues and binding to a high percentage of early stage pancreatic cancers. In preferred embodiments, the antibodies bind to pancreatic cancer mucins. The antibodies and fragments are of use for the detection and diagnosis of early stage pancreatic cancer. In preferred embodiments, the anti-pancreatic cancer antibodies can be used for immunoassay of serum samples, wherein the immunoassay can detect a marker for early stage pancreatic cancer in serum. More preferably, the serum is extracted with an organic phase, such as butanol, before immunoassay.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 12/537,803, filed Aug. 7, 2009, which was acontinuation-in-part of U.S. patent application Ser. No. 11/849,791 (nowabandoned), filed Sep. 4, 2007, which was a divisional of U.S. patentapplication Ser. No. 10/461,885 (now issued U.S. Pat. No. 7,282,567)filed Jun. 16, 2003, which claimed the benefit under 35 U.S.C. 119(e) ofU.S. Provisional Patent Application 60/388,314, filed Jun. 14, 2002.U.S. patent application Ser. No. 12/537,803 claimed the benefit under 35U.S.C. 119(e) of Provisional U.S. Patent Application Ser. Nos.61/087,463, filed Aug. 8, 2008, and 61/144,227, filed Jan. 13, 2009. Thepresent application claims the benefit under 35 U.S.C. 119(e) of U.S.Provisional Patent Application 61/297,303, filed Jan. 22, 2010;61/323,944, filed Apr. 14, 2010, 61/350,567, filed Jun. 2, 2010, and61/375,119, filed Aug. 19, 2010. The text of each priority applicationcited above is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This work was supported in part by NIH grant RO1-CA096924. The U.S.Government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the use of anti-pancreatic cancer antibodiesthat bind with high selectivity to pancreatic cancer cells to detectand/or diagnose pancreatic adenocarcinoma, preferably at the earlieststages of the disease. More preferably, antibody-based assays arecapable of detecting about 85% or more of pancreatic adenocarcinomas,with a false positive rate of 5% or less for healthy controls. Inparticular embodiments, the methods and compositions can be used todetect and/or diagnose pancreatic adenocarcinoma by screening serumsamples from subjects and preferably can detect 60% or more of stage Ipancreatic cancers and 80% or more of stage II cancers by serum sampleanalysis.

In preferred embodiments, the anti-pancreatic cancer antibody competesfor binding to pancreatic cancer mucin with a PAM4 antibody comprisingthe light chain variable region complementarity-determining region (CDR)sequences CDR1 (SASSSVSSSYLY, SEQ ID NO:1); CDR2 (STSNLAS, SEQ ID NO:2);and CDR3 (HQWNRYPYT, SEQ ID NO:3); and the heavy chain CDR sequencesCDR1 (SYVLH, SEQ ID NO:4); CDR2 (YINPYNDGTQYNEKFKG, SEQ ID NO:5) andCDR3 (GFGGSYGFAY, SEQ ID NO:6). Most preferably, the anti-pancreaticcancer antibody is a humanized PAM4 (hPAM4) antibody comprising thelight chain CDR sequences CDR1 (SASSSVSSSYLY, SEQ ID NO:1); CDR2(STSNLAS, SEQ ID NO:2); and CDR3 (HQWNRYPYT, SEQ ID NO:3); and the heavychain CDR sequences CDR1 (SYVLH, SEQ ID NO:4); CDR2 (YINPYNDGTQYNEKFKG,SEQ ID NO:5) and CDR3 (GFGGSYGFAY, SEQ ID NO:6), along with humanantibody framework (FR) and constant region sequences.

2. Related Art

Pancreatic cancer is a malignant growth of the pancreas that mainlyoccurs in the cells of the pancreatic ducts. This disease is the ninthmost common form of cancer, yet it is the fourth and fifth leading causeof cancer deaths in men and women, respectively. Cancer of the pancreasis almost always fatal, with a five-year survival rate that is less than3%.

The most common symptoms of pancreatic cancer include jaundice,abdominal pain, and weight loss, which, together with other presentingfactors, are nonspecific in nature. Thus, diagnosing pancreatic cancerat an early stage of tumor growth is often difficult and requiresextensive diagnostic work-up, often times including exploratory surgery.Endoscopic ultrasonography and computed tomography are the bestnoninvasive means available today for diagnosis of pancreatic cancer.However, reliable detection of small tumors, as well as differentiationof pancreatic cancer from focal pancreatitis, is difficult. The vastmajority of patients with pancreatic cancer are presently diagnosed at alate stage when the tumor has already extended outside of the capsule toinvade surrounding organs and/or has metastasized extensively. Gold etal., Crit. Rev. Oncology/Hematology, 39:147-54 (2001). Late detection ofthe disease is common, and early pancreatic cancer diagnosis is rare inthe clinical setting. This is significant, since late detection ofpancreatic cancer results in low survival rate.

Current treatment procedures available for pancreatic cancer have notled to a cure, or to a substantially improved survival time. Surgicalresection has been the only modality that offers a chance at survival.However, due to a large tumor burden, only 10% to 25% of patients arecandidates for “curative resection.” For those patients undergoing asurgical treatment, the five-year survival rate is still poor, averagingonly about 10%.

Early detection and diagnosis of pancreatic cancer, as well asappropriate staging of the disease, would provide an increased survivaladvantage. A number of laboratories have attempted to develop adiagnostic procedure based upon the release of a tumor-associated markerinto the bloodstream, as well as detection of the marker substancewithin biopsy specimens. The best previously-characterized tumorassociated marker for pancreatic cancer has been the immunoassay forCA19.9. Elevated levels of this sialylated Le^(a) epitope structure werefound in 70% of pancreatic cancer patients but were not found in any ofthe focal pancreatitis specimens examined. However, CA19.9 levels werefound to be elevated in a number of other malignant and benignconditions, and currently the assay cannot be used for diagnosis. Theassay is useful for monitoring, with continued increase in CA19.9 serumlevels after surgery being indicative of a poor prognosis. Many othermonoclonal antibodies (MAbs) have been reported with immunoassays fordiagnosis in varying stages of development. These include but are notlimited to DUPAN2, SPAN1, B72.3, Ia3, and various anti-CEA(carcinoembryonic antigen, or CEACAM5) antibodies.

Antibodies, in particular MAbs and engineered antibodies or antibodyfragments, have been tested widely and shown to be of value in detectionand treatment of various human disorders, including cancers, autoimmunediseases, infectious diseases, inflammatory diseases, and cardiovasculardiseases [Filpula and McGuire, Exp. Opin. Ther. Patents (1999) 9:231-245]. The clinical utility of an antibody or an antibody-derivedagent is primarily dependent on its ability to bind to a specifictargeted antigen associated with a particular disorder. Selectivity isvaluable for delivering a diagnostic or therapeutic agent, such asdrugs, toxins, cytokines, hormones, hormone antagonists, enzymes, enzymeinhibitors, oligonucleotides, growth factors, radionuclides,angiogenesis inhibitors or metals, to a target location during thedetection and treatment phases of a human disorder, particularly if thediagnostic or therapeutic agent is toxic to normal tissue in the body.Radiolabeled antibodies have been used with some success in numerousmalignancies, including ovarian cancer, colon cancer, medullary thyroidcancer, and lymphomas. This technology may also prove useful forpancreatic cancer. However, previously reported antibodies againstpancreatic cancer antigens have not been successfully employed to datefor the effective therapy or early detection and/or diagnosis ofpancreatic cancer.

There remains a need in the art for antibodies that exhibit highselectivity for pancreatic cancer and other types of cancers, comparedto normal pancreatic tissues and other normal tissues. In particular,there remains a need for antibodies that perform as a useful diagnosticand/or therapeutic tool for pancreatic cancer, preferably at theearliest stages of the disease, and that exhibit enhanced uptake attargeted antigens, decreased binding to constituents in the blood ofhealthy individuals and thereby also optimal protection of normaltissues and cells from toxic therapeutic agents when these areconjugated to such antibodies. Use of such antibodies to detectpancreatic cancer-associated antigens in body fluids, particularlyblood, can enable improved earlier diagnosis of this disease, so long asit differentiates well from benign diseases, and can also be used formonitoring response to therapy and potentially also to enhance prognosisby indicating disease burden.

SUMMARY

In various embodiments, the present invention concerns antibodies,antigen-binding antibody fragments and fusion proteins that bind topancreatic cancer cells, with little or no binding to normal ornon-neoplastic pancreatic cells. Preferably, the antibodies bind to theearliest stages of pancreatic cancer, with detection rates of about 54%for PanIN-1A, 75% for Pan1B and 86% for PanIN-2. More preferably, theantibodies bind to 80 to 90% or more of human invasive pancreaticadenocarcinoma, intraductal papillary mucinous neoplasia, PanIN-1A,PanIN-1B and PanIN-2 lesions. Most preferably, the antibodies candistinguish between early stage pancreatic cancer and non-malignantconditions such as pancreatitis. Such antibodies are of particular usefor early detection of cancer and differential diagnosis between earlystage pancreatic cancer and benign pancreatic conditions. In preferredembodiments, such antibodies are of use for in vivo or ex vivo analysisof samples from individuals suspected of having early stage pancreaticor certain other cancers. More preferably, the antibodies are of use fordetection and diagnosis of early stage pancreatic cancer by analysis ofserum samples.

The antibodies, antibody fragments or fusion proteins may be derived byimmunization and/or selection with mucin, and are preferably reactiveagainst mucin of human pancreatic cancer. Accordingly, the antibodies,antibody fragments and fusion proteins preferably bind to an antigenassociated with pancreatic cancer cells. More preferably, theantibodies, antibody fragments or fusion proteins may bind to a mucinexpressed in pancreatic cancer, such as MUC-1 or MUC-5.

In alternative embodiments, the antibodies, antibody fragments or fusionproteins may bind to synthetic peptide sequences, for example to phagedisplay peptides. Exemplary peptides that may bind to theanti-pancreatic cancer antibodies include, but are not limited to,WTWNITKAYPLP (SEQ ID NO:7) and ACPEWWGTTC (SEQ ID NO:8). Such syntheticpeptides may be linear or cyclic and may or may not compete withantibody binding to the endogenous pancreatic cancer antigen. Aminoacids in certain positions of the synthetic peptide sequences may beless critical for antibody binding than others. For example, in SEQ IDNO:7 the residues K, A and L at positions 7, 8 and 11 of the peptidesequence may be varied while still retaining antibody binding.Similarly, in SEQ ID NO:8 the threonine residues at positions 8 and 9 ofthe sequence may be varied, although substitution of the threonine atposition 9 may significantly affect antibody binding to the peptide.

Binding of the antibodies to a target pancreatic cancer antigen may beinhibited by treatment of the target antigen with reagents such asdithiothreitol (DTT) and/or periodate. Thus, binding of the antibodiesto a pancreatic cancer antigen may be dependent upon the presence ofdisulfide bonds and/or the glycosylation state of the target antigen. Inmore preferred embodiments, the epitope recognized by the subjectantibodies is not cross-reactive with other reported mucin-specificantibodies, such as the MA5 antibody, the CLH2-2 antibody and/or the45M1 antibody (see, e.g., Major et al., J Histochem Cytochem. 35:139-48,1987; Dion et al., Hybridoma 10:595-610, 1991).

The subject antibodies or fragments may be naked antibodies or fragmentsor preferably are conjugated to at least one therapeutic and/ordiagnostic agent for delivery of the agent to target tissues. Inalternative embodiments, the PAM4 antibodies or fragments may be part ofa bispecific fusion protein or antibody with a first binding site for atarget cell antigen and a second binding site for a hapten conjugated toa targetable construct. The targetable construct may in turn be attachedto at least one therapeutic and/or diagnostic agent, of use inpretargeting techniques.

In preferred embodiments, the subject antibody, antibody fragment orfusion protein comprises a murine, chimeric, humanized or human PAM4antibody or fragment, comprising the light chain variable region CDRsequences CDR1 (SASSSVSSSYLY, SEQ ID NO:1); CDR2 (STSNLAS, SEQ ID NO:2);and CDR3 (HQWNRYPYT, SEQ ID NO:3); and the heavy chain variable regionCDR sequences CDR1 (SYVLH, SEQ ID NO:4); CDR2 (YINPYNDGTQYNEKFKG, SEQ IDNO:5) and CDR3 (GFGGSYGFAY, SEQ ID NO:6).

Particular embodiments may concern compositions and methods of use ofmurine PAM4 antibodies, preferably comprising murine PAM4 variableregion sequences as disclosed in FIGS. 1A and 1B (SEQ ID NO:10 and SEQID NO:12). Such murine PAM4 antibodies or fragments may be of use in invitro or ex vivo diagnostic techniques, such as immunohistochemicalanalysis of tissue samples or immunoassay of body fluid samples fromsubjects suspected of having pancreatic cancer or other types of cancer.

Other particular embodiments may concern compositions and methods of useof chimeric PAM4 antibodies, comprising murine variable region sequencesattached to human antibody constant region sequences. Preferably thechimeric PAM4 antibodies or fragments comprise the cPAM4 variable regionsequences shown in FIGS. 2A and 2B (SEQ ID NO:13 and SEQ ID NO:14). Suchchimeric antibodies are less immunogenic in humans than murineantibodies, while retaining the antigen-binding specificities of theparent murine antibody. Chimeric antibodies are well known in the artfor use in diagnostic and/or therapeutic treatment of cancer.

Still other particular embodiments may concern compositions and methodsof use of humanized PAM4 antibodies or fragments thereof, comprising thecomplementarity-determining regions (CDRs) of a murine PAM4 MAb asdiscussed above and human antibody framework region (FR) and constantregion sequences. In a preferred embodiment, the FRs of the light andheavy chain variable regions of the humanized PAM4 antibody or fragmentthereof comprise at least one amino acid substituted from thecorresponding FRs of a murine PAM4 MAb. Still more preferred, thehumanized PAM4 antibody or fragment thereof may comprise at least aminoacid residue selected from amino acid residues 5, 27, 30, 38, 48, 66, 67and 69 of the murine PAM4 heavy chain variable region (FIG. 1B, SEQ IDNO:12) and/or at least one amino acid selected from amino acid residues21, 47, 59, 60, 85, 87 and 100 of the murine PAM4 light chain variableregion (FIG. 1A, SEQ ID NO:10). Most preferably, the humanized PAM4antibody or fragment thereof comprises the hPAM4 V_(H) amino acidsequence of FIG. 4A (SEQ ID NO:19) and the hPAM4 V_(k) amino acidsequence of FIG. 4B (SEQ ID NO:16).

In alternative embodiments, the anti-pancreatic cancer antibodies may bemurine, chimeric, humanized or human antibodies that bind to the sameantigenic determinant (epitope) as, or compete for binding to humanpancreatic mucin with, a chimeric PAM4 (cPAM4) antibody. As discussedbelow, the cPAM4 antibody is one that comprises the light chain variableregion CDR sequences CDR1 (SASSSVSSSYLY, SEQ ID NO:1); CDR2 (STSNLAS,SEQ ID NO:2); and CDR3 (HQWNRYPYT, SEQ ID NO:3); and the heavy chainvariable region CDR sequences CDR1 (SYVLH, SEQ ID NO:4); CDR2(YINPYNDGTQYNEKFKG, SEQ ID NO:5) and CDR3 (GFGGSYGFAY, SEQ ID NO:6).Antibodies that bind to the same antigenic determinant may be identifiedby a variety of techniques known in the art, such as by competitivebinding studies using the cPAM4 antibody as the competing antibody andhuman pancreatic mucin as the target antigen. (See, e.g., Example 1below.) Antibodies that block (compete for) binding to human pancreaticmucin of a cPAM4 antibody are referred to as cross-blocking antibodies.Preferably, such cross-blocking antibodies are prepared by immunizationwith extracts comprising human pancreatic cancer mucins.

Another embodiment is a cancer cell targeting diagnostic immunoconjugatecomprising an anti-pancreatic cancer antibody, antibody fragment orfusion protein that is bound to at least one diagnostic (or detection)agent.

Preferably, the diagnostic agent is selected from the group consistingof a radionuclide, a contrast agent, a fluorescent agent, achemiluminescent agent, a bioluminescent agent, a paramagnetic ion, anenzyme and a photoactive diagnostic agent. Still more preferred, thediagnostic agent is a radionuclide with an energy between 20 and 4,000keV or is a radionuclide selected from the group consisting of ¹¹⁰In,¹¹¹In, ¹⁷⁷Lu, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr,^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ³²P,¹¹C, ¹³N, ¹⁵O, ¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ^(52m)Mn, ⁵⁵Co, ⁷²As, ⁷⁵Br, ⁷⁶Br,^(82m)Rb, ⁸³Sr, or other gamma-, beta-, or positron-emitters. In aparticularly preferred embodiment, the diagnostic radionuclide ¹⁸F isused for labeling and PET imaging, as described in the Examples below.The ¹⁸F may be attached to an antibody, antibody fragment or peptide bycomplexation to a metal, such as aluminum, and binding of the ¹⁸F-metalcomplex to a chelating moiety that is conjugated to a targeting protein,peptide or other molecule.

Also preferred, the diagnostic agent is a paramagnetic ion, such aschromium (III), manganese (II), iron (III), iron (II), cobalt (II),nickel (II), copper (II), neodymium (III), samarium (III), ytterbium(III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III),holmium (III) and erbium (III), or a radiopaque material, such asbarium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid,iocetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide,iohexyl, iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid,ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, iotetricacid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid,ipodate, meglumine, metrizamide, metrizoate, propyliodone, and thallouschloride.

In still other embodiments, the diagnostic agent is a fluorescentlabeling compound selected from the group consisting of fluoresceinisothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin,o-phthaldehyde and fluorescamine, a chemiluminescent labeling compoundselected from the group consisting of luminol, isoluminol, an aromaticacridinium ester, an imidazole, an acridinium salt and an oxalate ester,or a bioluminescent compound selected from the group consisting ofluciferin, luciferase and aequorin. In another embodiment, thediagnostic immunoconjugates are used in intraoperative, endoscopic, orintravascular tumor diagnosis.

Another embodiment is a cancer cell-targeting therapeuticimmunoconjugate comprising an antibody or fragment thereof or fusionprotein bound to at least one therapeutic agent. Preferably, thetherapeutic agent is selected from the group consisting of aradionuclide, an immunomodulator, a hormone, a hormone antagonist, anenzyme, an oligonucleotide such as an anti-sense oligonucleotide or asiRNA, an enzyme inhibitor, a photoactive therapeutic agent, a cytotoxicagent such as a drug or toxin, an angiogenesis inhibitor and apro-apoptotic agent. In embodiments where more than one therapeuticagent is used, the therapeutic agents may comprise multiple copies ofthe same therapeutic agent or else combinations of different therapeuticagents.

In one embodiment, an oligonucleotide, such as an antisense molecule orsiRNA inhibiting bcl-2 expression as described in U.S. Pat. No.5,734,033 (the Examples section of which is incorporated herein byreference), may be conjugated to, or form the therapeutic agent portionof an immunoconjugate or antibody fusion protein. Alternatively, theoligonucleotide may be administered concurrently or sequentially with anaked or conjugated anti-pancreatic cancer antibody or antibodyfragment, such as a PAM4 antibody. In a preferred embodiment, theoligonucleotide is an antisense oligonucleotide that is directed againstan oncogene or oncogene product, such as bcl-2, p53, ras or otherwell-known oncogenes.

In a preferred embodiment, the therapeutic agent is a cytotoxic agent,such as a drug or a toxin. Also preferred, the drug is selected from thegroup consisting of nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, gemcitabine, triazenes, folic acid analogs,anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purineanalogs, antibiotics, enzyme inhibitors, epipodophyllotoxins, platinumcoordination complexes, vinca alkaloids, substituted ureas, methylhydrazine derivatives, adrenocortical suppressants, hormone antagonists,endostatin, taxols, camptothecins, SN-38, doxorubicins and theiranalogs, antimetabolites, alkylating agents, antimitotics,anti-angiogenic agents, tyrosine kinase inhibitors, mTOR inhibitors,heat shock protein (HSP90) inhibitors, proteosome inhibitors, HDACinhibitors, pro-apoptotic agents, methotrexate, CPT-11, and acombination thereof.

In another preferred embodiment, the therapeutic agent is a toxinselected from the group consisting of ricin, abrin, alpha toxin,saporin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A,pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonasexotoxin, and Pseudomonas endotoxin and combinations thereof. Or animmunomodulator selected from the group consisting of a cytokine, a stemcell growth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), a stem cell growthfactor, erythropoietin, thrombopoietin and a combinations thereof.

Alternatively, the therapeutic agent is an enzyme selected from thegroup consisting of malate dehydrogenase, staphylococcal nuclease,delta-V-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. Such enzymes may be used, for example, incombination with prodrugs that are administered in relatively non-toxicform and converted at the target site by the enzyme into a cytotoxicagent. In other alternatives, a drug may be converted into less toxicform by endogenous enzymes in the subject but may be reconverted into acytotoxic form by the therapeutic enzyme.

Other therapeutic agents include radionuclides such as ¹⁴C, ¹³N, ¹⁵O,³²P, ³³P, ⁴⁷Sc, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶²Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁷Ga, ⁷⁵Br,⁷⁵Se, ⁷⁵Se, ⁷⁶Br, ⁷⁷As, ⁷⁷Br, ^(80m)Br, ⁸⁹Sr, ⁹⁰Y, ⁹⁵Ru, ⁹⁷Ru, ⁹⁹Mo,^(99m)Tc, ^(103m)Rh, ¹⁰³Ru, ¹⁰⁵Rh, ¹⁰⁵Ru, ¹⁰⁷Hg, ¹⁰⁹Pd, ¹⁰⁹Pt, ¹¹¹Ag,¹¹¹In, ^(113m)In, ¹¹⁹Sb, ^(121m)Te, ^(122m)Te, ¹²⁵I, ^(125m)Te, ¹²⁶I,¹³¹I, ¹³³I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵²Dy, ¹⁵³Sm, ¹⁶¹Ho, ¹⁶¹Tb, ¹⁶⁵Tm,¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁶⁹Er, ¹⁶⁹Yb, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re,^(189m)Os, ¹⁸⁹Re, ¹⁹²Ir, ¹⁹⁴Ir, ¹⁹⁷Pt, ¹⁹⁸Au, ¹⁹⁹Au, ²⁰¹Tl, ²⁰³Hg,²¹¹At, ²¹¹Bi, ²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²¹⁵Po, ²¹⁷At, ²¹⁹Rn, ²²¹Fr,²²³Ra, ²²⁴Ac, ²²⁵Ac or ²⁵⁵Fm.

Also contemplated are multivalent, multispecific antibodies or fragmentsthereof comprising at least one binding site having an affinity toward aPAM4 target antigen and one or more hapten binding sites having affinitytowards hapten molecules. Preferably, the antibody or fragment thereofis a chimeric, humanized or fully human antibody or fragment thereof.The hapten molecule may be conjugated to a targetable construct fordelivery of one or more therapeutic and/or diagnostic agents. In certainpreferred embodiments, the multivalent antibodies or fragments thereofmay be prepared by the dock-and-lock (DNL) technique, as described inthe Examples below. An exemplary DNL construct incorporating hPAM4antibody fragments is designated TF10, as described below.

Also contemplated is a bispecific antibody or fragment thereofcomprising at least one binding site with an affinity toward a PAM4target antigen and at least one binding site with an affinity toward atargetable construct/conjugates selected from the group consisting of:

-   DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH₂ (IMP 271);-   DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂ (IMP 277);-   DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂ (IMP 288);-   DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂ (IMP 0281);-   NOTA-ITC benzyl-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ (IMP 449);-   NODA-Ga-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ (IMP 460);-   NOTA-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ (IMP 461);-   NOTA-D-Asp-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ (IMP 462);-   NOTA-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ (IMP 465);-   C-NETA-succinyl-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ (IMP 467);-   S-NETA-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ (IMP 469); and-   L-NETA-succinyl-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ (IMP 470)    which is capable of carrying at least one diagnostic and/or    therapeutic agent. Other targetable constructs suitable for use are    disclosed, for example, in U.S. Pat. Nos. 6,576,746; 6,962,702;    7,052,872; 7,138,103; 7,172,751; 7,405,320; 7,597,876; 7,563,433;    and U.S. patent application Ser. Nos. 12/343,655, filed Dec. 24,    2008, 12/433,212, filed Apr. 30, 2009, 12/485,998, filed Jun. 17,    2009, 12/541,527, filed Aug. 14, 2009 and 12/958,889, filed Dec. 1,    2010, the Examples section of each of which is incorporated herein    by reference.

Other embodiments concern fusion proteins comprising at least twoanti-pancreatic cancer antibodies and fragments thereof as describedherein. Alternatively, the fusion protein or fragment thereof maycomprise at least one first anti-pancreatic cancer antibody or fragmentthereof and at least one second MAb or fragment thereof. Preferably, thesecond MAb binds to a tumor-associated antigen, for example selectedfrom the group consisting of CA19.9, DUPAN2, SPAN1, Nd2, B72.3, CC49,CEA (CEACAM5), CEACAM6, Le^(a), the Lewis antigen Le(y), CSAp,insulin-like growth factor (IGF), epithelial glycoprotein-1 (EGP-1),epithelial glycoprotein-2 (EGP-2), CD-80, placental growth factor(P1GF), carbonic anhydrase IX, tenascin, IL-6, HLA-DR, CD40, CD74 (e.g.,milatuzumab), CD138 (syndecan-1), MUC-1, MUC-2, MUC-3, MUC-4, MUC-5ac,MUC-16, MUC-17, TAG-72, EGFR, platelet-derived growth factor (PDGF),angiogenesis factors (e.g., VEGF and P1GF), products of oncogenes (e.g.,bcl-2, Kras, p53), cMET, HER2/neu, and antigens associated with gastriccancer and colorectal cancer. The antibody fusion protein or fragmentsthereof may further comprise at least one diagnostic and/or therapeuticagent.

Also described herein are DNA sequences comprising a nucleic acidencoding an anti-pancreatic cancer antibody, fusion protein,multispecific antibody, bispecific antibody or fragment thereof asdescribed herein. Other embodiments concern expression vectors and/orhost cells comprising the antibody-encoding DNA sequences. In certainpreferred embodiments, the host cell may be an Sp2/0 cell linetransformed with a mutant Bcl-2 gene, for example with a triple mutantBcl-2 gene (T69E, S70E, S87E), that has been adapted to celltransformation and growth in serum free medium. (See, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930; and 7,608,425, the Examples section of eachof which is incorporated herein by reference.)

Another embodiment concerns methods of delivering a diagnostic ortherapeutic agent, or a combination thereof, to a target comprising (i)providing a composition that comprises an anti-pancreatic cancerantibody or fragment, such as a PAM4 antibody or fragment, conjugated toat least one diagnostic and/or therapeutic agent and (ii) administeringto a subject in need thereof the diagnostic or therapeutic conjugate ofany one of the antibodies, antibody fragments or fusion proteins claimedherein.

Also contemplated is a method of delivering a diagnostic agent, atherapeutic agent, or a combination thereof to a target, comprising: (a)administering to a subject any one of the multivalent, multispecific orbispecific antibodies or fragments thereof that have an affinity towarda PAM4 antigen and comprise one or more hapten binding sites; (b)waiting a sufficient amount of time for antibody that does not bind tothe PAM4 antigen to clear the subject's blood stream; and (c)administering to said subject a carrier molecule comprising a diagnosticagent, a therapeutic agent, or a combination thereof, that binds to abinding site of the antibody. Preferably, the carrier molecule binds tomore than one binding site of the antibody.

Described herein is a method for diagnosing or treating cancer,comprising: (a) administering to a subject any one of the multivalent,multispecific antibodies or fragments thereof claimed herein that havean affinity toward a PAM4 antigen and comprise one or more haptenbinding sites; (b) waiting a sufficient amount of time for an amount ofthe non-bound antibody to clear the subject's blood stream; and (c)administering to said subject a carrier molecule comprising a diagnosticagent, a therapeutic agent, or a combination thereof, that binds to abinding site of the antibody. In a preferred embodiment the cancer ispancreatic cancer. Also preferred, the method can be used forintraoperative identification of diseased tissues, endoscopicidentification of diseased tissues, or intravascular identification ofdiseased tissues.

Another embodiment is a method of treating a malignancy in a subjectcomprising administering to said subject a therapeutically effectiveamount of an antibody or fragment thereof that binds to a PAM4 antigen,optionally conjugated to at least one therapeutic agent. The antibody orfragment thereof may alternatively be a naked antibody or fragmentthereof. In more preferred embodiments, the antibody or fragment isadministered either before, simultaneously with, or after administrationof another therapeutic agent as described above.

Contemplated herein is a method of diagnosing a malignancy in a subject,particularly a pancreatic cancer, comprising (a) administering to saidsubject a diagnostic conjugate comprising an antibody or fragmentthereof that binds to a PAM4 antigen, wherein said MAb or fragmentthereof is conjugated to at least one diagnostic agent, and (b)detecting the presence of labeled antibody bound to pancreatic cancercells or other malignant cells, wherein binding of the antibody isdiagnostic for the presence of pancreatic cancer or another malignancy.In preferred embodiments, the antibody or fragment binds to pancreaticcancer and not to normal pancreatic tissue, pancreatitis or othernon-malignant conditions. In less preferred embodiments, the antibody orfragment binds at a significantly higher level to cancer cells than tonon-malignant cells, allowing differential diagnosis of cancer fromnon-malignant conditions. In a most preferred embodiment, the diagnosticagent may be an F-18 labeled molecule that is detected by PET imaging.

In more preferred embodiments, the use of anti-pancreatic cancerantibodies, such as the hPAM4 antibody, allows the detection and/ordiagnosis of pancreatic cancer with high specificity and sensitivity atthe earliest stages of malignant disease. Preferably, the diagnosticantibody or fragment is capable of labeling at least 70%, morepreferably at least 80%, more preferably at least 90%, more preferablyat least 95%, most preferably about 100% of well differentiated,moderately differentiated and poorly differentiated pancreatic cancerand 90% or more of invasive pancreatic adenocarcinomas. Theanti-pancreatic cancer antibody of use is preferably capable ofdetecting 85% or more of PanIN-1A, PanIN-1B, PanIN-2, IPMN and MCNprecursor lesions. Most preferably, immunoassays using theanti-pancreatic cancer antibody are capable of detecting 89% or more oftotal PanIN, 86% or more of IPMN, and 92% or more of MCN.

An alternative embodiment is a method of detecting PAM4 antigen and/ordiagnosing pancreatic cancer in an individual by in vitro analysis ofblood, plasma or serum samples. Preferably, the sample is subjected toan organic phase extraction, using an organic solvent such as butanol,before it is processed for immunodetection using an anti-pancreaticcancer antibody, such as a PAM4 antibody. Following organic phaseextraction, the extracted aqueous phase is analyzed for the presence ofPAM4 antigen in the sample, using any of a variety of immunoassaytechniques known in the art, such as ELISA, sandwich immunoassay, solidphase RIA, and similar techniques. Surprisingly, the organic phaseextraction results in the removal of an inhibitor of PAM4 binding to itstarget antigen, allowing detection of the PAM4 target antigen in freshserum samples. More surprisingly, using the in vitro analysis techniquesdescribed herein, serum samples may be analyzed to detect and/ordiagnose pancreatic cancer in a subject at the earliest stages ofpancreatic adenocarcinoma. These unexpected results provide the firstserum-based assay technique that is diagnostic for the presence of earlystage pancreatic cancer.

Another embodiment is a method of treating a cancer cell in a subjectcomprising administering to said subject a composition comprising anaked antibody or fragment thereof or a naked antibody fusion protein orfragment thereof that binds to a PAM4 antigen. Preferably, the methodfurther comprises administering a second naked antibody or fragmentthereof selected from the group consisting of CA19.9, DUPAN2, SPAN1,Nd2, B72.3, CC49, anti-CEA, anti-CEACAM6, anti-EGP-1, anti-EGP-2,anti-Le^(a), antibodies defined by the Lewis antigen Le(y), andantibodies against CSAp, MUC-1, MUC-2, MUC-3, MUC-4, MUC-Sac, MUC-16,MUC-17, TAG-72, EGFR, CD40, HLA-DR, CD74, CD138, angiogenesis factors(e.g., VEGF and placenta-like growth factor (P1GF), insulin-like growthfactor (IGF), tenascin, platelet-derived growth factor, IL-6, productsof oncogenes, cMET, and HER2/neu.

Still other embodiments concern a method of diagnosing a malignancy in asubject comprising (i) performing an in vitro diagnosis assay on aspecimen from said subject with a composition comprising an antibody orfragment thereof that binds to a PAM4 antigen; and

(ii) detecting the presence of antibody or fragment bound to malignantcells in the specimen. Preferably, the malignancy is a cancer. Stillpreferred, the cancer is pancreatic cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Variable region cDNA sequences and the deduced amino acidsequences of the murine PAM4 antibody. FIG. 1A shows the DNA (SEQ IDNO:9) and amino acid (SEQ ID NO:10) sequences of the murine PAM4 Vk.FIG. 1B shows the DNA (SEQ ID NO:11) and amino acid (SEQ ID NO:12)sequences of the murine PAM4 V_(H). Amino acid sequences encoded by thecorresponding DNA sequences are given as one-letter codes below thenucleotide sequence. Numbering of the nucleotide sequence is on theright side. The amino acid residues in the CDR regions are shown in boldand underlined. Kabat's Ig molecule numbering is used for amino acidresidues as shown by the numbering above the amino acid residues. Theamino acid residues numbered by a letter are the insertion residuesdefined by Kabat numbering scheme. The insertion residues have the samepreceding digits as that of the previous residue. For example, residues82, 82A, 82B, and 82C in FIG. 1B are indicated as 82, A, B, and C,respectively.

FIG. 2. Amino acid sequences of the chimeric PAM4 (cPAM4) heavy andlight chain variable regions expressed in Sp2/0 cells. FIG. 2A shows theamino acid sequence (SEQ ID NO:13) of the cPAM4 Vk. FIG. 2B shows theamino acid sequence (SEQ ID NO:14) of the cPAM4 V_(H). The sequences aregiven as one letter codes. The amino acid residues in the CDR regionsare shown in bold and underlined. The numbering of amino acids is thesame as in FIG. 1.

FIG. 3. Alignment of the amino acid sequences of heavy and light chainvariable regions of a human antibody, PAM4 and hPAM4. FIG. 3A shows theV_(k) amino acid sequence alignment of the human antibody Walker (SEQ IDNO:15) with PAM4 (SEQ ID NO:10) and hPAM4 (SEQ ID NO:16), and FIG. 3Bshows the V_(H) amino acid sequence alignment of the human antibody Wil2(FR1-3) (SEQ ID NO:17) and NEWM (FR4) (SEQ ID NO:18) with PAM4 (SEQ IDNO:12) and hPAM4 (SEQ ID NO:19). Dots indicate the residues of PAM4 thatare identical to the corresponding residues of the human or humanizedantibodies. Boxed regions represent the CDR regions. Both N- andC-terminal residues (underlined) of hPAM4 are fixed by the stagingvectors used. Kabat's Ig molecule number scheme is used to number theresidues as in FIG. 1.

FIG. 4. DNA and amino acid sequences of the humanized PAM4 (hPAM4) heavyand light chain variable regions expressed in Sp2/0 cells. FIG. 4A showsthe DNA (SEQ ID NO:20) and amino acid (SEQ ID NO:16) sequences of thehPAM4 V_(k) and FIG. 4B shows the DNA (SEQ ID NO:21) and amino acid (SEQID NO:19) sequences of the hPAM4 V_(H). Numbering of the nucleotidesequence is on the right side. Amino acid sequences encoded by thecorresponding DNA sequences are given as one-letter codes. The aminoacid residues in the CDR regions are shown in bold and underlined.Kabat's Ig molecule numbering scheme is used for amino acid residues asin FIG. 1A and FIG. 1B.

FIG. 5. Binding activity of humanized PAM4 antibody, hPAM4, as comparedto the chimeric PAM4, cPAM4. hPAM4 is shown by diamonds and cPAM4 isshown by closed circles. Results indicate comparable binding activity ofthe hPAM4 antibody and cPAM4 when competing with ¹²⁵I-cPAM4 binding toCaPan1 antigens.

FIG. 6. PET/CT fusion images for a patient with inoperable metastaticpancreatic cancer treated with fractionated ⁹⁰Y-hPAM4 plus gemcitabine,before therapy (left side) and post-therapy (right side). The circleindicates the location of the primary lesion, which shows a significantdecrease in PET/CT intensity following therapy.

FIG. 7. 3D PET images for a patient with inoperable metastaticpancreatic cancer treated with fractionated ⁹⁰Y-hPAM4 plus gemcitabine,before therapy (left side) and post-therapy (right side). Arrows pointto the locations of the primary lesion (on right) and metastases (onleft), each of which shows a significant decrease in PET image intensityafter therapy with radiolabeled hPAM4 plus gemcitabine.

FIG. 8. In vivo imaging of tumors using an ¹¹¹In-labeled diHSG peptide(IMP 288) with or without pretargeting TF10 bispecific anti-pancreaticcancer mucin antibody. FIG. 8A—mice showing location of tumors (arrow).FIG. 8B shows the detected tumors with ¹¹¹In-labeled IMP 288 in thepresence (above) or absence (below) of TF10 bispecific antibody.

FIG. 9. Exemplary binding curves for TF10, PAM4-IgG, PAM4-F(ab′)₂ and amonovalent bsPAM4 chemical conjugate (PAM4-Fab′ x anti-DTPA-Fab′).Binding to target mucin antigen was measured by ELISA assay.

FIG. 10. Immunoscintigraphy of CaPan1 human pancreatic cancer xenografts(˜0.25 g). (A) An image of mice that were injected with bispecific TF10(80 μg, 5.07×10⁻¹⁰ mol) followed 16 h later by administration of¹¹¹In-IMP-288 (30 μCi, 5.07×10⁻¹¹ mol). The image was taken 3 h later.The intensity of the image background was increased to match theintensity of the image obtained when ¹¹¹In-IMP-288 was administeredalone (30 μCi, 5.07×10⁻¹¹ mol). (B) No targeting was observed in micegiven ¹¹¹In-IMP-288 alone. (C) An image of mice that were given¹¹¹In-DOTA-PAM4-IgG (20 μCi, 50 μg) with imaging done 24 h later.Although tumors are visible, considerable background activity is stillpresent at this time point.

FIG. 11. Extended biodistribution of ¹¹¹In-DOTA-PAM4-IgG (20 μCi, 50 μg)and TF10-pretargeted ¹¹¹In-IMP-288 (80 μg, 5.07×10⁻¹⁰ mol TF10 followed16 h later with 30 μCi, 5.07×10⁻¹¹ mol ¹¹¹In-IMP-288) in nude micebearing CaPan1 human pancreatic cancer xenografts (mean tumorweight+/−SD, 0.28+/−0.21 and 0.10+/−0.06 g for the pretargeting and IgGgroups of animals, respectively)

FIG. 12. Therapeutic activity of a single treatment of established (˜0.4cm³) CaPan1 tumors with 0.15 mCi of ⁹⁰Y-hPAM4 IgG, or 0.25 or 0.50 mCiof TF10-pretargeted ⁹⁰Y-IMP-288.

FIG. 13. Effect of gemcitabine potentiation of PT-RAIT therapy.

FIG. 14. Effect of combination of cetuximab with gemcitabine andPT-RAIT.

FIG. 15. Differential diagnosis of pancreatic cancer using PAM4-basedimmunoassay. The red line shows the cutoff level selected for a positiveresult, based on ROC analysis.

FIG. 16. Frequency distribution of PAM4 antigen in patient sera fromhealthy volunteers and individuals with varying stages of pancreaticcancer.

FIG. 17. ROC curve for PAM4 serum immunoassay.

FIG. 18. Accuracy of the PAM4-immunoassay was determined to be within10% of the nominal concentrations examined at or above the cutoff valueof 2.40 units/mL. A linear trend was calculated with an equation ofy=0.965x+0.174, and goodness of fit r²=0.999.

FIG. 19. Frequency distribution of PAM4-reactive antigen in patient seraby stage of disease. Cutoff value=2.4 units/mL (red line). The medianvalues (units/mL) are shown for each study group.

FIG. 20. Receiver Operator Characteristics (ROC) curve for theperformance of the PAM4-based immunoassay; pancreatic adenocarcinoma vs.healthy adults. Values for the area under the curves (AUC) and 95%confidence limits are provided.

DETAILED DESCRIPTION Definitions

Unless otherwise specified, “a” or “an” means one or more.

As used herein, “about” means plus or minus 10%. For example, “about100” would include any number between 90 and 110.

As described herein, the term “PAM4 antibody” includes murine, chimeric,humanized and human PAM4 antibodies. In preferred embodiments, the PAM4antibody or antigen-binding fragment thereof comprises the CDR sequencesof SEQ ID NO:1 to SEQ ID NO:6.

As used herein, a “PAM4 antigen” is an antigen bound by a PAM4 antibody.In preferred embodiments, treatment of PAM4 antigen with DTT orperiodate inhibits or prevents binding of the PAM4 antibody. In morepreferred embodiments, the PAM4 antigen is an epitope of a mucinexpressed by a pancreatic cancer cell, such as MUC-1 or MUC-5.

As used herein, an “anti-pancreatic cancer antibody” is an antibody thatexhibits the same diagnostic, therapeutic and binding characteristics asthe PAM4 antibody. In preferred embodiments, the “anti-pancreatic cancerantibody” binds to the same epitope as the PAM4 antibody.

A “non-endocrine pancreatic cancer” generally refers to cancers arisingfrom the exocrine pancreatic glands. The term excludes pancreaticinsulinomas and includes pancreatic carcinoma, pancreaticadenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma andgiant cell carcinoma and precursor lesions such as pancreaticintra-epithelial neoplasia (PanIN), mucinous cyst neoplasms (MCN) andintrapancreatic mucinous neoplasms (IPMN), which are neoplastic but notyet malignant. The terms “pancreatic cancer” and “non-endocrinepancreatic cancer” are used interchangeably herein.

An antibody, as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment.

An antibody fragment is a portion of an antibody such as F(ab)₂, Fab′,Fab, Fv, sFv and the like. Regardless of structure, an antibody fragmentbinds with the same antigen that is recognized by the full-lengthantibody. The term “antibody fragment” also includes isolated fragmentsconsisting of the variable regions of antibodies, such as the “Fv”fragments consisting of the variable regions of the heavy and lightchains and recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“scFvproteins”).

A naked antibody is an antibody or fragment thereof that is notconjugated to a therapeutic or diagnostic agent. Generally, the Fcportion of the antibody molecule provides effector functions, such ascomplement-mediated cytotoxicity (CDC) and ADCC (antibody-dependentcellular cytotoxicity), which set mechanisms into action that may resultin cell lysis. However, the Fc portion may not be required fortherapeutic function, with other mechanisms, such as signaling-inducedapoptosis, coming into play. Naked antibodies include both polyclonaland monoclonal antibodies, as well as fusion proteins and certainrecombinant antibodies, such as chimeric, humanized or human antibodies.

A chimeric antibody is a recombinant protein that contains the variabledomains including the complementarity determining regions (CDRs) of anantibody derived from one species, preferably a rodent antibody, whilethe constant domains of the antibody molecule are derived from those ofa human antibody. For veterinary applications, the constant domains ofthe chimeric antibody may be derived from that of other species, such asa cat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a rodent antibody, are transferred fromthe heavy and light variable chains of the rodent antibody into humanheavy and light variable domains (e.g., framework region sequences). Theconstant domains of the antibody molecule are derived from those of ahuman antibody. In certain embodiments, a limited number of frameworkregion amino acid residues from the parent (rodent) antibody may besubstituted into the human antibody framework region sequences.

A human antibody is, e.g., an antibody obtained from transgenic micethat have been “engineered” to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain loci are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous murine heavy chain and light chain loci. Thetransgenic mice can synthesize human antibodies specific for particularantigens, and the mice can be used to produce human antibody-secretinghybridomas. Methods for obtaining human antibodies from transgenic miceare described by Green et al., Nature Genet. 7:13 (1994), Lonberg etal., Nature 368:856 (1994), and Taylor et al., Int. Immun. 6:579 (1994).A fully human antibody also can be constructed by genetic or chromosomaltransfection methods, as well as phage display technology, all of whichare known in the art. See for example, McCafferty et al., Nature348:552-553 (1990) for the production of human antibodies and fragmentsthereof in vitro, from immunoglobulin variable domain gene repertoiresfrom unimmunized donors. In this technique, antibody variable domaingenes are cloned in-frame into either a major or minor coat protein geneof a filamentous bacteriophage, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. In this way, the phage mimics some of the properties of theB cell. Phage display can be performed in a variety of formats, forreview, see e.g. Johnson and Chiswell, Current Opinion in StructuralBiology 3:5564-571 (1993). Human antibodies may also be generated by invitro activated B cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275, theExamples sections of which are incorporated herein by reference.

A therapeutic agent is a compound, molecule or atom which isadministered separately, concurrently or sequentially with an antibodymoiety or conjugated to an antibody moiety, i.e., antibody or antibodyfragment, or a subfragment, and is useful in the treatment of a disease.Examples of therapeutic agents include antibodies, antibody fragments,cytotoxic agents, drugs, toxins, nucleases, hormones, immunomodulators,pro-apoptotic agents, anti-angiogenic agents, boron compounds,photoactive agents or dyes and radioisotopes. Therapeutic agents of useare described in more detail below.

A diagnostic agent is a molecule, atom or other detectable moiety whichmay be administered conjugated to an antibody moiety or targetableconstruct and is useful in detecting or diagnosing a disease by locatingcells containing the target antigen. Useful diagnostic agents include,but are not limited to, radioisotopes, dyes, contrast agents,fluorescent compounds or molecules and enhancing agents (e.g.,paramagnetic ions) for magnetic resonance imaging (MRI) or positronemission tomography (PET) scanning. Preferably, the diagnostic agentsare selected from the group consisting of radioisotopes, enhancingagents for use in magnetic resonance or PET imaging, and fluorescentcompounds. In order to load an antibody component with radioactivemetals, paramagnetic ions or other diagnostic cations, it may benecessary to react it with a reagent having a long tail to which areattached a multiplicity of chelating groups for binding the ions. Such atail can be a polymer such as a polylysine, polysaccharide, or otherderivatized or derivatizable chain having pendant groups to which can bebound chelating groups such as, e.g., ethylenediaminetetraacetic acid(EDTA), diethylenetriaminepentaacetic acid (DTPA), DOTA, NOTA, NETA,porphyrins, polyamines, crown ethers, bis-thiosemicarbazones,polyoximes, and like groups known to be useful for this purpose.Chelates are coupled to the antibodies using standard chemistries. Thechelate is normally linked to the antibody by a group which enablesformation of a bond to the molecule with minimal loss ofimmunoreactivity and minimal aggregation and/or internal cross-linking.Other, more unusual, methods and reagents for conjugating chelates toantibodies are disclosed in U.S. Pat. No. 4,824,659, the Examplessection of which is incorporated herein by reference. Particularlyuseful metal-chelate combinations include 2-benzyl-DTPA and itsmonomethyl and cyclohexyl analogs, used with diagnostic isotopes in thegeneral energy range of 60 to 4,000 keV, such as ¹²⁵I, ¹³¹I, ¹²³I, ¹²⁴I,⁶²Cu, ⁶⁴Cu, ¹⁸F, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Br, forradioimaging. The same chelates, when complexed with non-radioactivemetals, such as manganese, iron and gadolinium are useful for MRI, whenused along with the antibodies of the invention. Macrocyclic chelatessuch as NOTA (1,4,7-triazacyclononane-N,N′,N″-triacetic acid), DOTA(1,4,7,10-tetraazacyclododecanetetraacetic acid), and TETA(p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid) are of usewith a variety of metals and radiometals, most particularly withradionuclides of gallium, yttrium and copper, respectively. Suchmetal-chelate complexes can be made very stable by tailoring the ringsize to the metal of interest. Other ring-type chelates such asmacrocyclic polyethers, which are of interest for stably bindingnuclides, such as ²²³Ra for radioimmunotherapy (RAIT) are encompassed bythe invention. More recently, techniques of general utility for labelingvirtually any molecule with an ¹⁸F atom of use in PET imaging have beendescribed in U.S. Pat. No. 7,563,433, the Examples section of which isincorporated herein by reference.

An immunoconjugate is an antibody, antibody fragment or antibody fusionprotein conjugated to at least one therapeutic and/or diagnostic agent.The diagnostic agent and/or therapeutic agent are as defined above.

An expression vector is a DNA molecule comprising a gene that isexpressed in a host cell. Typically, gene expression is placed under thecontrol of certain regulatory elements, including constitutive orinducible promoters, tissue-specific regulatory elements and enhancers.Such a gene is said to be “operably linked to” the regulatory elements.

A recombinant host may be any prokaryotic or eukaryotic cell thatcontains either a cloning vector or expression vector. This term alsoincludes those prokaryotic or eukaryotic cells, as well as transgenicanimals, that have been genetically engineered to contain the clonedgene(s) in the chromosome or genome of the host cell. Suitable mammalianhost cells include myeloma cells, such as SP2/0 cells, and NS0 cells, aswell as Chinese Hamster Ovary (CHO) cells, hybridoma cell lines andother mammalian host cell useful for expressing antibodies. Alsoparticularly useful to express mAbs and other fusion proteins are Sp2/0cells transfected with an apoptosis inhibitor, such as a Bel-EEE gene,and adapted to grow and be further transfected in serum free conditions,as described in U.S. Pat. Nos. 7,531,327; 7,537,930; and 7,608,425, theExamples section of each of which is incorporated herein by reference.

PAM4 Antibodies

Various embodiments of the invention concern antibodies that react withvery high selectivity with pancreatic cancer as opposed to normal orbenign pancreatic tissues. The anti-pancreatic cancer antibodies andfragments thereof are preferably raised against a crude mucinpreparation from a tumor of the human pancreas, although partiallypurified or even purified mucins may be utilized. A non-limiting exampleof such antibodies is the PAM4 antibody.

The murine PAM4 (mPAM4) antibody was developed by employing pancreaticcancer mucin derived from the xenografted RIP-1 human pancreaticcarcinoma as immunogen. (Gold et al., Int. J. Cancer, 57:204-210, 1994.)As discussed below, antibody cross-reactivity and immunohistochemicalstaining studies indicate that the PAM4 antibody recognizes a unique andnovel epitope on a target pancreatic cancer antigen. Immunohistochemicalstaining studies, such as those described in Example 2, have shown thatthe PAM4 MAb binds to an antigen expressed by breast, pancreas and othercancer cells, with limited binding to normal human tissue; however, thehighest expression is usually by pancreatic cancer cells. Thus, the PAM4antibodies are relatively specific to pancreatic cancer andpreferentially bind pancreatic cancer cells. The PAM4 antibody isreactive with a target epitope which can be internalized. This epitopeis expressed primarily by antigens associated with pancreatic cancer andnot with focal pancreatitis or normal pancreatic tissue. Binding of PAM4antibody to the PAM4 epitope is inhibited by treatment of the antigenwith DIT or periodate. Localization and therapy studies using aradiolabeled PAM4 MAb in animal models have demonstrated tumor targetingand therapeutic efficacy.

The PAM4 antibodies bind to PAM4 antigen, which is expressed by manyorgans and tumor types, but is preferentially expressed in pancreaticcancer cells. Studies with a PAM4 MAb, such as in Example 3, indicatethat the antibody exhibits several important properties, which make it agood candidate for clinical diagnostic and therapeutic applications. ThePAM4 antigen provides a useful target for diagnosis and therapy ofpancreatic and other cancers. The PAM4 antibody apparently recognizes anepitope of a pancreatic cancer antigen that is distinct from theepitopes recognized by non-PAM4 anti-pancreatic cancer antibodies(CA19.9, DUPAN2, SPAN1, Nd2, CEACAM5, B72.3, anti-Le^(a), and otheranti-Lewis antigens).

Surprisingly, the Examples below indicate that the PAM4 antigen ispresent in detectable concentrations in serum of patients with veryearly stage pancreatic cancer. Also surprisingly, it appears that anendogenous inhibitor of PAM4 antibody binding to the PAM4 antigen ispresent in fresh human serum. The inhibitor is removed by long-termfrozen storage of serum samples, or by organic phase extraction of freshserum. These unexpected results provide the basis of a relativelynon-invasive, early detection test for pancreatic cancer, using blood,serum or plasma samples.

For therapeutic use, antibodies suitable for use in combination orconjunction with PAM4 antibodies include, for example, the antibodiesCA19.9, DUPAN2, SPAN1, Nd2, B72.3, CC49, anti-CEA, anti-CEACAM6,anti-Le^(a), anti-HLA-DR, anti-CD40, anti-CD74, anti-CD138, andantibodies defined by the Lewis antigen Le(y), or antibodies againstcolon-specific antigen-p (CSAp), MUC-1, MUC-2, MUC-3, MUC-4, MUC-5ac,MUC-16, MUC-17, EGP-1, EGP-2, HER2/neu, EGFR, angiogenesis factors(e.g., VEGF and P1GF), insulin-like growth factor (IGF), tenascin,platelet-derived growth factor, and IL-6, as well as products ofoncogenes (bc1-2, Kras, p53), cMET, and antibodies against tumornecrosis substances, such as described in patents by Epstein et al.(U.S. Pat. Nos. 6,071,491, 6,017,514, 5,019,368 and 5,882,626). Suchantibodies would be useful for complementing PAM4 antibodyimmunodetection and immunotherapy methods. These and other therapeuticagents could act synergistically with anti-pancreatic cancer antibodies,such as PAM4 antibody, when administered before, together with or afteradministration of PAM4 antibody.

In therapeutic applications, antibodies that are agonistic orantagonistic to immunomodulators involved in effector cell functionagainst tumor cells could also be useful in combination with PAM4antibodies alone or in combination with other tumor-associatedantibodies, one example being antibodies against CD40. Todryk et al., J.Immunol. Methods, 248:139-147 (2001); Turner et al., J. Immunol,166:89-94 (2001). Also of use are antibodies against markers or productsof oncogenes (e.g., bc1-2, Kras, p53, cMET), or antibodies againstangiogenesis factors, such as VEGFR and placenta-like growth factor(P1GF).

The availability of another PAM4-like antibody that binds to a differentepitope of the PAM4 antigen is important for the development of adouble-determinant enzyme-linked immunosorbent assay (ELISA), of use fordetecting a PAM4 antigen in clinical samples. ELISA experiments aredescribed in Example 1 and 5.

The murine, chimeric, humanized and fully human PAM4 antibodies andfragments thereof described herein are exemplary of anti-pancreaticcancer antibodies of use for diagnostic and/or therapeutic methods. TheExamples below disclose a preferred embodiment of the construction anduse of a humanized PMA4 antibody. Because non-human monoclonalantibodies can be recognized by the human host as a foreign protein, andrepeated injections can lead to harmful hypersensitivity reactions,humanization of a murine antibody sequences can reduce the adverseimmune response that patients may experience. For murine-basedmonoclonal antibodies, this is often referred to as a Human Anti-MouseAntibody (HAMA) response. Preferably some human residues in theframework regions of the humanized anti-pancreatic cancer antibody orfragments thereof are replaced by their murine counterparts. It is alsopreferred that a combination of framework sequences from two differenthuman antibodies is used for V_(H). The constant domains of the antibodymolecule are derived from those of a human antibody.

Antibody Preparation

Monoclonal antibodies for specific antigens may be obtained by methodsknown to those skilled in the art. See, for example, Kohler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991) (hereinafter “Coligan”). Briefly, anti-pancreatic cancer MAbs canbe obtained by injecting mice with a composition comprising mucins frompancreatic cancer, such as the PAM4 antigen, verifying the presence ofantibody production by removing a serum sample, removing the spleen toobtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells toproduce hybridomas, cloning the hybridomas, selecting positive cloneswhich produce antibodies to PAM4 antigen, culturing the clones thatproduce antibodies to PAM4 antigen, and isolating anti-pancreatic cancerantibodies from the hybridoma cultures.

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques toproduce chimeric or humanized antibodies. Chimerization of murineantibodies and antibody fragments are well known to those skilled in theart. The use of antibody components derived from chimerized monoclonalantibodies reduces potential problems associated with the immunogenicityof murine constant regions.

General techniques for cloning murine immunoglobulin variable domainsare described, for example, by the publication of Orlandi et al., ProcNat'l Acad. Sci. USA 86: 3833 (1989), incorporated herein by reference.In general, the V_(K) (variable light chain) and V_(H) (variable heavychain) sequences for murine antibodies can be obtained by a variety ofmolecular cloning procedures, such as RT-PCR, 5′-RACE, and cDNA libraryscreening. Specifically, the V_(H) and V_(K) sequences of the murinePAM4 MAb were cloned by PCR amplification from the hybridoma cells byRT-PCR, and their sequences determined by DNA sequencing. To confirmtheir authenticity, the cloned V_(K) and V_(H) genes can be expressed incell culture as a chimeric Ab as described by Orlandi et al., (ProcNatl. Acad. Sci., USA, 86: 3833, 1989).

In a preferred embodiment, a chimerized PAM4 antibody or antibodyfragment comprises the complementarity-determining regions (CDRs) andframework regions (FR) of a murine PAM4 MAb and the light and heavychain constant regions of a human antibody, wherein the CDRs of thelight chain variable region of the chimerized PAM4 comprises CDR1comprising an amino acid sequence of SASSSVSSSYLY (SEQ ID NO:1); CDR2comprising an amino acid sequence of STSNLAS (SEQ ID NO:2); and CDR3comprising an amino acid sequence of HQWNRYPYT (SEQ ID NO:3); and theCDRs of the heavy chain variable region of the chimerized PAM4 MAbcomprises CDR1 comprising an amino acid sequence of SYVLH (SEQ ID NO:4);CDR2 comprising an amino acid sequence of YINPYNDGTQYNEKFKG (SEQ IDNO:5) and CDR3 comprising an amino acid sequence of GFGGSYGFAY (SEQ IDNO:6). The use of antibody components derived from chimerized monoclonalantibodies reduces potential problems associated with the immunogenicityof murine constant regions.

Humanization of murine antibodies and antibody fragments is also wellknown to those skilled in the art. Techniques for producing humanizedMAbs are disclosed, for example, by Carter et al., Proc Nat'l Acad. Sci.USA 89: 4285 (1992), Singer et al., J. Immun. 150: 2844 (1992), Mountainet al. Biotechnol Genet Eng Rev. 10:1 (1992), and Coligan at pages10.19.1-10.19.11, each of which is incorporated herein by reference. Forexample, humanized monoclonal antibodies may be produced by transferringmurine complementary determining regions from heavy and light variablechains of the mouse immunoglobulin into a human variable domain, andthen, substituting human residues in the framework regions of the murinecounterparts. The use of human framework region sequences, in additionto human constant region sequences, further reduces the chance ofinducing HAMA reactions.

Based on the PAM4 variable region sequences, obtained as describedabove, a humanized PAM4 antibody can be designed and constructed asdescribed by Leung et al. (Mol. Immunol. 32: 1413 (1995)), incorporatedherein by reference. Example 1 describes the humanization processutilized for construction of the hPAM4 MAb.

The nucleotide sequences of the primers used to prepare the hPAM4antibodies are discussed in Example 1, below. In a preferred embodiment,a humanized PAM4 antibody or antibody fragment comprises the light andheavy chain CDR sequences (SEQ ID NO:1 to SEQ ID NO:6) disclosed above.Also preferred, the FRs of the light and heavy chain variable regions ofthe humanized antibody comprise at least one amino acid substituted fromsaid corresponding FRs of the murine PAM4 MAb.

A fully human antibody, e.g., human PAM4 can be obtained from atransgenic non-human animal. See, e.g., Mendez et al., Nature Genetics,15: 146-156 (1997); U.S. Pat. No. 5,633,425. For example, a humanantibody can be recovered from a transgenic mouse possessing humanimmunoglobulin loci. The mouse humoral immune system is humanized byinactivating the endogenous immunoglobulin genes and introducing humanimmunoglobulin loci. The human immunoglobulin loci are exceedinglycomplex and comprise a large number of discrete segments which togetheroccupy almost 0.2% of the human genome. To ensure that transgenic miceare capable of producing adequate repertoires of antibodies, largeportions of human heavy- and light-chain loci must be introduced intothe mouse genome. This is accomplished in a stepwise process beginningwith the formation of yeast artificial chromosomes (YACs) containingeither human heavy- or light-chain immunoglobulin loci in germlineconfiguration. Since each insert is approximately 1 Mb in size, YACconstruction requires homologous recombination of overlapping fragmentsof the immunoglobulin loci. The two YACs, one containing the heavy-chainloci and one containing the light-chain loci, are introduced separatelyinto mice via fusion of YAC-containing yeast spheroblasts with mouseembryonic stem cells. Embryonic stem cell clones are then microinjectedinto mouse blastocysts. Resulting chimeric males are screened for theirability to transmit the YAC through their germline and are bred withmice deficient in murine antibody production. Breeding the twotransgenic strains, one containing the human heavy-chain loci and theother containing the human light-chain loci, creates progeny whichproduce human antibodies in response to immunization. However, thesetechniques are not limiting and other methods known in the art forproducing human antibodies, such as the use of phage display, may alsobe utilized to produce human anti-pancreatic cancer antibodies.

Antibodies can be produced by cell culture techniques using methodsknown in the art. In one example transfectoma cultures are adapted toserum-free medium. For production of humanized antibody, cells may begrown as a 500 ml culture in roller bottles using HSFM. Cultures arecentrifuged and the supernatant filtered through a 0.2 μm membrane. Thefiltered medium is passed through a protein-A column (1×3 cm) at a flowrate of 1 ml/min. The resin is then washed with about 10 column volumesof PBS and protein A-bound antibody is eluted from the column with 0.1 Mglycine buffer (pH 3.5) containing 10 mM EDTA. Fractions of 1.0 ml arecollected in tubes containing 10 μl of 3 M Tris (pH 8.6), and proteinconcentrations determined from the absorbance at 280/260 nm. Peakfractions are pooled, dialyzed against PBS, and the antibodyconcentrated, for example, with a Centricon 30 filter (Amicon, Beverly,Mass.). The antibody concentration is determined by ELISA and itsconcentration adjusted to about 1 mg/ml using PBS. Sodium azide, 0.01%(w/v), is conveniently added to the sample as preservative.

Antibodies can be isolated and purified from hybridoma cultures by avariety of well-established techniques. Such isolation techniquesinclude affinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

Anti-pancreatic cancer MAbs can be characterized by a variety oftechniques that are well-known to those of skill in the art. Forexample, the ability of an antibody to bind to the PAM4 antigen can beverified using an indirect enzyme immunoassay, flow cytometry analysis,ELISA or Western blot analysis.

Antibody Fragments

Antibody fragments are antigen binding portions of an antibody, such asF(ab′)₂, Fab′, F(ab)₂, Fab, Fv, sFv, scFv and the like. Antibodyfragments which recognize specific epitopes can be generated by knowntechniques. F(ab)₂ fragments, for example, can be produced by pepsindigestion of the antibody molecule. These and other methods aredescribed, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647 and references contained therein. Also, see Nisonoff et al.,Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119(1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page 422(Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4. Alternatively, Fab′ expression libraries can beconstructed (Huse et al., 1989, Science, 246:1274-1281) to allow rapidand easy identification of monoclonal Fab′ fragments with the desiredspecificity.

A single chain Fv molecule (scFv) comprises a V_(L) domain and a V_(H)domain. The V_(L) and V_(H) domains associate to form a target bindingsite. These two domains are further covalently linked by a peptidelinker (L). A scFv molecule is denoted as either V_(L)-L-V_(H) if theV_(L) domain is the N-terminal part of the scFv molecule, or asV_(H)-L-V_(L) if the V_(H) domain is the N-terminal part of the scFvmolecule. Methods for making scFv molecules and designing suitablepeptide linkers are described in U.S. Pat. No. 4,704,692, U.S. Pat. No.4,946,778, R. Raag and M. Whitlow, “Single Chain Fvs.” FASEB Vol 9:73-80(1995) and R. E. Bird and B. W. Walker, Single Chain Antibody VariableRegions, TIBTECH, Vol 9: 132-137 (1991).

Other antibody fragments, for example single domain antibody fragments,are known in the art and may be used in the claimed constructs. Singledomain antibodies (VHH) may be obtained, for example, from camels,alpacas or llamas by standard immunization techniques. (See, e.g.,Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods281:161-75, 2003; Maass et al., J Immunol Methods 324:13-25, 2007). TheVHH may have potent antigen-binding capacity and can interact with novelepitopes that are inaccessible to conventional V_(H)-V_(L) pairs.(Muyldermans et al., 2001). Alpaca serum IgG contains about 50% camelidheavy chain only IgG antibodies (HCAbs) (Maass et al., 2007). Alpacasmay be immunized with known antigens, such as TNF-α, and VHHs can beisolated that bind to and neutralize the target antigen (Maass et al.,2007). PCR primers that amplify virtually all alpaca VHH codingsequences have been identified and may be used to construct alpaca VHHphage display libraries, which can be used for antibody fragmentisolation by standard biopanning techniques well known in the art (Maasset al., 2007).

An antibody fragment can also be prepared by proteolytic hydrolysis of afull-length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full-length antibodies by conventionalmethods. For example, an antibody fragment can be produced by enzymaticcleavage of antibodies with pepsin to provide an approximate 100 Kdfragment denoted F(ab')₂. This fragment can be further cleaved using athiol reducing agent, and optionally a blocking group for the sulthydrylgroups resulting from cleavage of disulfide linkages, to produce anapproximate 50 Kd Fab′ monovalent fragment. Alternatively, an enzymaticcleavage using papain produces two monovalent Fab fragments and an Fcfragment directly.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Antibody Fusion Proteins and Multivalent Antibodies

Fusion proteins comprising the anti-pancreatic cancer antibodies ofinterest can be prepared by a variety of conventional procedures,ranging from glutaraldehyde linkage to more specific linkages betweenfunctional groups. The antibodies and/or antibody fragments thatcomprise the fusion proteins described herein are preferably covalentlybound to one another, directly or through a linker moiety, through oneor more functional groups on the antibody or fragment, e.g., amine,carboxyl, phenyl, thiol, or hydroxyl groups. Various conventionallinkers in addition to glutaraldehyde can be used, e.g., diisocyanates,diiosothiocyanates, bis(hydroxysuccinimide)esters, carbodiimides,maleimidehydroxy succinimide esters, and the like.

A simple method for producing fusion proteins is to mix the antibodiesor fragments in the presence of glutaraldehyde. The initial Schiff baselinkages can be stabilized, e.g., by borohydride reduction to secondaryamines. A diiosothiocyanate or carbodiimide can be used in place ofglutaraldehyde as a non-site-specific linker. In one embodiment, anantibody fusion protein comprises an anti-pancreatic cancer MAb, orfragment thereof, wherein the MAb binds to the PAM4 antigen. This fusionprotein and fragments thereof preferentially bind pancreatic cancercells. This monovalent, monospecific MAb is useful for direct targetingof an antigen, where the MAb is attached to a therapeutic agent, adiagnostic agent, or a combination thereof, and the protein isadministered directly to a patient.

The fusion proteins may instead comprise at least two anti-pancreaticcancer MAbs that bind to distinct epitopes of the PAM4 antigen. Forexample, the MAbs can produce antigen specific diabodies, triabodies andtetrabodies, which are multivalent but monospecific to the PAM4 antigen.The non-covalent association of two or more scFv molecules can formfunctional diabodies, triabodies and tetrabodies. Monospecific diabodiesare homodimers of the same scFv, where each scFv comprises the V_(H)domain from the selected antibody connected by a short linker to theV_(L) domain of the same antibody. A diabody is a bivalent dimer formedby the non-covalent association of two scFvs, yielding two Fv bindingsites. A triabody results from the formation of a trivalent trimer ofthree scFvs, yielding three binding sites, and a tetrabody is atetravalent tetramer of four scFvs, resulting in four binding sites.Several monospecific diabodies have been made using an expression vectorthat contains a recombinant gene construct comprisingV_(H)1-linker-V_(L)1. See Holliger et al., Proc Natl. Acad. Sci. USA 90:6444-6448 (1993); Atwell et al., Molecular Immunology 33: 1301-1302(1996); Holliger et al., Nature Biotechnology 15: 632-631 (1997);Helfrich et al., Int J Cancer 76: 232-239 (1998); Kipriyanov et al., IntJ Cancer 77: 763-772 (1998); Holiger et al., Cancer

Research 59: 2909-2916 (1999)). Methods of constructing scFvs aredisclosed in U.S. Pat. No. 4,946,778 (1990) and U.S. Pat. No. 5,132,405(1992), the Examples section of each of which is incorporated herein byreference. Methods of producing multivalent, monospecific antibodiesbased on scFv are disclosed in U.S. Pat. No. 5,837,242 (1998), U.S. Pat.No. 5,844,094 (1998) and WO-98/44001 (1998), the Examples section ofeach of which is incorporated herein by reference. The multivalent,monospecific antibody fusion protein binds to two or more of the sametype of epitopes that can be situated on the same antigen or on separateantigens. The increased valency allows for additional interaction,increased affinity, and longer residence times. These antibody fusionproteins can be utilized in direct targeting systems, where the antibodyfusion protein is conjugated to a therapeutic agent, a diagnostic agent,or a combination thereof, and administered directly to a patient in needthereof.

A preferred embodiment is a multivalent, multispecific antibody orfragment thereof comprising one or more antigen binding sites having anaffinity toward a PAM4 target epitope and one or more additional bindingsites for other epitopes associated with pancreatic cancer. This fusionprotein is multispecific because it binds at least two differentepitopes, which can reside on the same or different antigens. Forexample, the fusion protein may comprise more than one antigen bindingsite, the first with an affinity toward a PAM4 antigen epitope and thesecond with an affinity toward another target antigen such as TAG-72 orCEA. Another example is a bispecific antibody fusion protein which maycomprise a CA19.9 MAb (or fragment thereof) and a PAM4 MAb (or fragmentthereof). Such a fusion protein will have an affinity toward CA19.9 aswell as the PAM4 antigen. The antibody fusion proteins and fragmentsthereof can be utilized in direct targeting systems, where the antibodyfusion protein is conjugated to a therapeutic agent, a diagnostic agent,or a combination thereof, and administered directly to a patient in needthereof.

Another preferred embodiment is a multivalent, multispecific antibodycomprising at least one binding site having affinity toward a PAM4target epitope and at least one hapten binding site having affinitytowards hapten molecules. For example, a bispecific fusion protein maycomprise the 679 MAb (or fragment thereof) and the PAM4 MAb (or fragmentthereof). The monoclonal 679 antibody binds with high affinity tomolecules containing the tri-peptide moiety histamine succinyl glycyl(HSG). Such a bispecific PAM4 antibody fusion protein can be prepared,for example, by obtaining a F(ab′)₂ fragment from 679, as describedabove. The interchain disulfide bridges of the 679 F(ab)₂ fragment aregently reduced with DTT, taking care to avoid light-heavy chain linkage,to form Fab′-SH fragments. The SH group(s) is (are) activated with anexcess of bis-maleimide linker(1,1′-(methylenedi-4,1-phenylene)b-is-maleimide). The PAM4 MAb isconverted to Fab′-SH and then reacted with the activated 679 Fab′-SHfragment to obtain a bispecific antibody fusion protein. Bispecificantibody fusion proteins such as this one can be utilized in affinityenhancing systems, where the target antigen is pretargeted with thefusion protein and is subsequently targeted with a diagnostic ortherapeutic agent attached to a carrier moiety (targetable construct)containing one or more HSG haptens. In alternative preferredembodiments, a DNL-based hPAM4-679 construct, such as TF10, may beprepared and used as described in the Examples below.

Bispecific antibodies can be made by a variety of conventional methods,e.g., disulfide cleavage and reformation of mixtures of whole IgG or,preferably F(ab′)₂ fragments, fusions of more than one hybridoma to formpolyomas that produce antibodies having more than one specificity, andby genetic engineering. Bispecific antibody fusion proteins have beenprepared by oxidative cleavage of Fab′ fragments resulting fromreductive cleavage of different antibodies. This is advantageouslycarried out by mixing two different F(ab′)₂ fragments produced by pepsindigestion of two different antibodies, reductive cleavage to form amixture of Fab′ fragments, followed by oxidative reformation of thedisulfide linkages to produce a mixture of F(ab′)₂ fragments includingbispecific antibody fusion proteins containing a Fab′ portion specificto each of the original epitopes. General techniques for the preparationof antibody fusion proteins may be found, for example, in Nisonoff etal., Arch Biochem Biophys. 93: 470 (1961), Hammerling et al., J Exp Med.128: 1461 (1968), and U.S. Pat. No. 4,331,647.

More selective linkage can be achieved by using a heterobifunctionallinker such as maleimidehydroxysuccinimide ester. Reaction of the esterwith an antibody or fragment will derivatize amine groups on theantibody or fragment, and the derivative can then be reacted with, e.g.,an antibody Fab fragment having free sulfhydryl groups (or, a largerfragment or intact antibody with sulfhydryl groups appended thereto by,e.g., Traut's Reagent). Such a linker is less likely to crosslink groupsin the same antibody and improves the selectivity of the linkage.

It is advantageous to link the antibodies or fragments at sites remotefrom the antigen-binding sites. This can be accomplished by, e.g.,linkage to cleaved interchain sulfhydryl groups, as noted above. Anothermethod involves reacting an antibody having an oxidized carbohydrateportion with another antibody that has at lease one free amine function.This results in an initial Schiff base linkage, which is preferablystabilized by reduction to a secondary amine, e.g., by borohydridereduction, to form the final composite. Such site-specific linkages aredisclosed, for small molecules, in U.S. Pat. No. 4,671,958, and forlarger addends in U.S. Pat. No. 4,699,784, the Examples section of eachof which is incorporated herein by reference.

ScFvs with linkers greater than 12 amino acid residues in length (forexample, 15- or 18-residue linkers) allow interactions between the V_(H)and V_(L) domains on the same chain and generally form a mixture ofmonomers, dimers (termed diabodies) and small amounts of higher massmultimers, (Kortt et al., Eur J. Biochem. (1994) 221: 151-157). ScFvswith linkers of 5 or less amino acid residues, however, prohibitintramolecular pairing of the V_(H) and V_(L) domains on the same chain,forcing pairing with V_(H) and V_(L) domains on a different chain.Linkers between 3- and 12-residues form predominantly dimers (Atwell etal., Protein Engineering (1999) 12: 597-604). With linkers between 0 and2 residues, trimeric (termed triabodies), tetrameric (termedtetrabodies) or higher oligomeric structures of scFvs are formed;however, the exact patterns of oligomerization appear to depend on thecomposition as well as the orientation of the V-domains, in addition tothe linker length.

More recently, a novel technique for construction of mixtures ofantibodies, antibody fragments and/or other effector moieties invirtually any combination has been described in U.S. Pat. Nos.7,550,143; 7,521,056; 7,534,866; 7,527,787; and 7,666,400, the Examplessection of each of which is incorporated herein by reference. Thetechnique, known generally as dock-and-lock (DNL) involves theproduction of fusion proteins that comprise at their N- or C-terminalends one of two complementary peptide sequences, called dimerization anddocking domain (DDD) and anchoring domain (AD) sequences. In preferredembodiments, the DDD sequences are derived from the regulatory subunitsof cAMP-dependent protein kinase and the AD sequence is derived from thesequence of A-kinase anchoring protein (AKAP). The DDD sequences formdimers that bind to the AD sequence, which allows formation of trimers,tetramers, hexamers or any of a variety of other complexes. By attachingeffector moieties, such as antibodies or antibody fragments, to the DDDand AD sequences, complexes may be formed of any selected combination ofantibodies or antibody fragments. The DNL complexes may be covalentlystabilized by formation of disulfide bonds or other linkages.

Known Antibodies

Bispecific antibodies comprising the antigen-binding variable regionsequences of any known anti-TAA antibody may be utilized, including butnot limited to hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No.7,251,164), hA19 (U.S. Pat. No. 7,109,304), hIMMU31 (U.S. Pat. No.7,300,655), hLL1 (U.S. Pat. No. 7,312,318,), hLL2 (U.S. Pat. No.7,074,403), hMu-9 (U.S. Pat. No. 7,387,773), hL243 (U.S. Pat. No.7,612,180), hMN-14 (U.S. Pat. No. 6,676,924), hRS7 (U.S. Pat. No.7,238,785), hMN-3 (U.S. Pat. No. 7,541,440), hMN-15 (U.S. patentapplication Ser. No. 12/846,062, filed Jul. 29, 2010) and hR1 (U.S.patent application Ser. No. 12/772,645, filed Mar. 12, 2010) theExamples section of each cited patent or application incorporated hereinby reference.

Other antibodies of use may be commercially obtained from a wide varietyof known sources. For example, a variety of antibody secreting hybridomalines are available from the American Type Culture Collection (ATCC,Manassas, Va.). A large number of antibodies against various diseasetargets, including but not limited to tumor-associated antigens, havebeen deposited at the ATCC and/or have published variable regionsequences and are available for use in the claimed methods andcompositions. See, e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164;7,074,403; 7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803;7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598;6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018;6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244;6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533;6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625;6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580;6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226;6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206;6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681;6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,15; 6,716,966;6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355;6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852;6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279;6,596,852; 6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618;6,545,130; 6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227;6,518,404; 6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408;6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356;6,455,044; 6,455,040, 6,451,310; 6,444,206′ 6,441,143; 6,432,404;6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091;6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654;6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244;6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393;6,254,868; 6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289;6,077,499; 5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554;5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953,5,525,338, the Examples section of each of which is incorporated hereinby reference. These are exemplary only and a wide variety of otherantibodies and their hybridomas are known in the art. The skilledartisan will realize that antibody sequences or antibody-secretinghybridomas against almost any disease-associated antigen may be obtainedby a simple search of the ATCC, NCBI and/or USPTO databases forantibodies against a selected disease-associated target of interest. Theantigen binding domains of the cloned antibodies may be amplified,excised, ligated into an expression vector, transfected into an adaptedhost cell and used for protein production, using standard techniqueswell known in the art.

Pretargeting

Bispecific or multispecific antibodies may be utilized in pre-targetingtechniques. Pre-targeting is a multistep process originally developed toresolve the slow blood clearance of directly targeting antibodies, whichcontributes to undesirable toxicity to normal tissues such as bonemarrow. With pre-targeting, a radionuclide or other therapeutic agent isattached to a small delivery molecule (targetable construct ortargetable conjugate) that is cleared within minutes from the blood. Apre-targeting bispecific or multispecific antibody, which has bindingsites for the targetable construct as well as a target antigen, isadministered first, free antibody is allowed to clear from circulationand then the targetable construct is administered.

Pre-targeting methods are well known in the art, for example, asdisclosed in Goodwin et al., U.S. Pat. No. 4,863,713; Goodwin et al., J.Nucl. Med. 29:226, 1988; Hnatowich et al., J. Nucl. Med. 28:1294, 1987;Oehr et al., J. Nucl. Med. 29:728, 1988; Klibanov et al., J. Nucl. Med.29:1951, 1988; Sinitsyn et al., J. Nucl. Med. 30:66, 1989; Kalofonos etal., J. Nucl. Med. 31:1791, 1990; Schechter et al., Int. J. Cancer48:167, 1991; Paganelli et al., Cancer Res. 51:5960, 1991; Paganelli etal., Nucl. Med. Commun. 12:211, 1991; U.S. Pat. No. 5,256,395; Stickneyet al., Cancer Res. 51:6650, 1991; Yuan et al., Cancer Res. 51:3119,1991; U.S. Pat. No. 6,077,499; U.S. Pat. No. 6,090,381; U.S. Pat. No.6,472,511; and U.S. Pat. No. 6,962,702.

A pre-targeting method of treating or diagnosing a disease or disorderin a subject may be provided by: (1) administering to the subject abispecific antibody or antigen binding antibody fragment; (2) optionallyadministering to the subject a clearing composition, and allowing thecomposition to clear the antibody from circulation; and (3)administering to the subject the targetable construct, containing one ormore chelated or chemically bound therapeutic or diagnostic agents. Thetechnique may also be utilized for antibody dependent enzyme prodrugtherapy (ADEPT) by administering an enzyme conjugated to a targetableconstruct, followed by a prodrug that is converted into active form bythe enzyme.

Antibody Use for Treatment and Diagnosis

Certain embodiments concern methods of diagnosing or treating amalignancy in a subject, comprising administering to the subject ananti-pancreatic cancer MAb, fusion protein or fragment thereof, whereinthe MAb, fusion protein or fragment is bound to at least one diagnosticand/or therapeutic agent. Also preferred is a method for diagnosing ortreating cancer, comprising administering to a subject a multivalent,multispecific antibody or fragment thereof comprising one or moreantigen binding sites toward a PAM4 antigen and one or more haptenbinding sites, waiting a sufficient amount of time for non-boundantibody to clear the subject's blood stream; and then administering tothe subject a carrier molecule comprising a diagnostic agent, atherapeutic agent, or a combination thereof, that binds to thehapten-binding site of the localized antibody. In a more preferredembodiment, the cancer is a non-endocrine pancreatic cancer.

The use of MAbs for in vitro diagnosis is well-known. See, for example,Carlsson et al., Bio/Technology 7 (6): 567 (1989). For example, MAbs canbe used to detect the presence of a tumor-associated antigen in tissuefrom biopsy samples. MAbs also can be used to measure the amount oftumor-associated antigen in clinical fluid samples using techniques suchas radioimmunoassay, enzyme-linked immunosorbent assay, and fluorescenceimmunoassay. In vitro and in vivo methods of diagnosis are discussed infurther detail below.

Conjugates of tumor-targeted MAbs and toxins can be used to selectivelykill cancer cells in vivo (Spalding, Bio/Technology 9(8): 701 (1991);Goldenberg, Scientific American Science & Medicine 1(1): 64 (1994)). Forexample, therapeutic studies in experimental animal models havedemonstrated the anti-tumor activity of antibodies carrying cytotoxicradionuclides. (Goldenberg et al., Cancer Res. 41: 4354 (1981), Cheunget al., J. Nat'l Cancer Inst. 77: 739 (1986), and Senekowitsch et al.,J. Nucl. Med. 30: 531 (1989)). In a preferred embodiment, the conjugatecomprises a ⁹⁰Y-labeled hPAM4 antibody. The conjugate may optionally beadministered in conjunction with one or more other therapeutic agents.In a preferred embodiment, ⁹⁰Y-labeled hPAM4 is administered togetherwith gemcitabine or 5-fluorouracil to a patient with pancreatic cancer.In a further preferred embodiment, ⁹⁰Y is conjugated to a DOTA chelatefor attachment to hPAM4. In a still further preferred embodiment, the⁹⁰Y-DOTA-hPAM4 is combined with gemcitabine in fractionated dosescomprising a treatment cycle, such as with repeated, lower, less-toxicdoses of gemcitabine combined with lower, fractionated doses of⁹⁰Y-DOTA-hPAM4.

As tolerated, repeated cycles of this fractionated dose schedule areindicated. By way of example, 4 weekly doses of 200 mg/m² of gemcitabineare combined with three weekly doses of 8 mg/m² of ⁹⁰Y-DOTA-hPAM4, withthe latter commencing in the second week of gemcitabine administration,constitutes a single therapy cycle. Still other doses, higher and lowerof each component, may constitute a fractionated dose, which isdetermined by conventional means of assessing hematopoietic toxicity(see, e.g., U.S. Pat. Nos. 6,649,352; 7,112,409; 7,279,289; 7,465,551),since myelosuppressive effects of both agents can be cumulative. Askilled physician in such therapy interventions can adjust these dosesbased on the patient's bone marrow status and general health statusbased on many factors, including prior exposure to myelosuppressivetherapeutic agents. These principles can also apply to combinations ofradiolabeled hPAM4 with other therapeutic agents, includingradiosensitizing drugs such as 5-fluorouracil and cisplatin.

Chimeric, humanized and human antibodies and fragments thereof have beenused for in vivo therapeutic and diagnostic methods. Accordinglycontemplated is a method of delivering a diagnostic or therapeuticagent, or a combination thereof, to a target comprising (i) providing acomposition that comprises an anti-pancreatic cancer antibody orfragment thereof, such as a chimeric, humanized or human PAM4 antibody,conjugated to at least one diagnostic and/or therapeutic agent and (ii)administering to a subject the diagnostic or therapeutic antibodyconjugate. In a preferred embodiment, the anti-pancreatic cancerantibodies and fragments thereof are humanized or fully human.

Another embodiment concerns a method for treating a malignancycomprising administering a naked anti-pancreatic cancer antibody,antibody fragment or fusion protein, such as a PAM4 antibody, eitheralone or in conjunction with one or more other therapeutic agents. Theother therapeutic agent may be added before, simultaneously with orafter the antibody. In a preferred embodiment, the therapeutic agent isgemcitabine, and in a more preferred embodiment, gemcitabine is givenwith the hPAM4 radioconjugate in a fractionated dose schedule at lowerdoses than the conventional 800-1,000 mg/m² doses of gemcitabine givenweekly for 6 weeks. For example, when combined with fractionatedtherapeutic doses of ⁹⁰Y-PAM4, repeated fractionated doses intended tofunction as a radio sensitizing agent of 200-380 mg/m² gemcitabine areinfused. The skilled artisan will realize that the antibodies, fusionproteins and/or fragments thereof described and claimed herein may beadministered with any known or described therapeutic agent, includingbut not limited to heat shock protein 90 (Hsp90).

In another form of multimodal therapy, subjects receive immunoconjugatesin conjunction with standard cancer chemotherapy. For example, “CVB”(1.5 g/m² cyclophosphamide, 200-400 mg/m² etoposide, and 150-200 mg/m²carmustine) is a regimen used to treat non-Hodgkin's lymphoma. Patti etal., Eur. J. Haematol. 51: 18 (1993). Other suitable combinationchemotherapeutic regimens are well-known to those of skill in the art.See, for example, Freedman et al., “Non-Hodgkin's Lymphomas,” in CANCERMEDICINE, VOLUME 2, 3rd Edition, Holland et al. (eds.), pages 2028-2068(Lea & Febiger 1993). As an illustration, first generationchemotherapeutic regimens for treatment of intermediate-gradenon-Hodgkin's lymphoma (NHL) include C-MOPP (cyclophosphamide,vincristine, procarbazine and prednisone) and CHOP (cyclophosphamide,doxorubicin, vincristine, and prednisone). A useful second generationchemotherapeutic regimen is m-BACOD (methotrexate, bleomycin,doxorubicin, cyclophosphamide, vincristine, dexamethasone andleucovorin), while a suitable third generation regimen is MACOP-B(methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisone,bleomycin and leucovorin). Additional useful drugs include phenylbutyrate, bendamustine, and bryostatin-1.

The present invention contemplates the administration of anti-pancreaticcancer antibodies and fragments thereof, including fusion proteins andfragments thereof, alone, as a naked antibody or antibody fragment, oradministered as a multimodal therapy. Preferably, the antibody is ahumanized or fully human PAM4 antibody or fragment thereof. Multimodaltherapies further include immunotherapy with a naked anti-pancreaticcancer antibody supplemented with administration of other antibodies inthe form of naked antibodies, fusion proteins, or as immunoconjugates.For example, a humanized or fully human PAM4 antibody may be combinedwith another naked antibody, or a humanized PAM4 or other antibodyconjugated to an isotope, one or more chemotherapeutic agents,cytokines, toxins or a combination thereof. For example, the presentinvention contemplates treatment of a naked or conjugated PAM4 antibodyor fragments thereof before, in combination with, or after otherpancreatic tumor associated antibodies such as CA19.9, DUPAN2, SPAN1,Nd2, B72.3, CC49, anti-Le^(a) antibodies, and antibodies to other Lewisantigens (e.g., Le(y)), as well as antibodies against carcinoembryonicantigen (CEA or CEACAM5), CEACAM6, colon-specific antigen-p (CSAp),MUC-1, MUC-2, MUC-3, MUC-4, MUC-5ac, MUC-16, MUC-17, HLA-DR, CD40, CD74,CD138, HER2/neu, EGFR, EGP-1, EGP-2, angiogenesis factors (e.g., VEGF,P1GF), insulin-like growth factor (IGF), tenascin, platelet-derivedgrowth factor, and IL-6, as well as products of oncogenes (e.g., bcl-2,Kras, p53), cMET, and antibodies against tumor necrosis substances.

These solid tumor antibodies may be naked or conjugated to, inter alia,drugs, toxins, isotopes, radionuclides or immunomodulators. Manydifferent antibody combinations may be constructed and used in eithernaked or conjugated form. Alternatively, different naked antibodycombinations may be employed for administration in combination withother therapeutic agents, such as a cytotoxic drug or with radiation,given consecutively, simultaneously, or sequentially.

Administration of the antibodies and their fragments can be effected byintravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, intrapleural, intrathecal, perfusion through a regionalcatheter, or direct intralesional injection. When administering theantibody by injection, the administration may be by continuous infusionor by single or multiple boluses.

The immunoconjugate of the present invention can be formulated forintravenous administration via, for example, bolus injection orcontinuous infusion. Preferably, the antibody of the present inventionis infused over a period of less than about 4 hours, and morepreferably, over a period of less than about 3 hours. For example, thefirst 25-50 mg could be infused within 30 minutes, preferably even 15min, and the remainder infused over the next 2-3 hrs. Formulations forinjection can be presented in unit dosage form, e.g., in ampoules or inmulti-dose containers, with an added preservative. The compositions cantake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic conjugate. Control releasepreparations can be prepared through the use of polymers to complex oradsorb the immunoconjugate. For example, biocompatible polymers includematrices of poly(ethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebacic acid.Sherwood et al., Bio/Technology 10: 1446 (1992). The rate of release ofan immunoconjugate or antibody from such a matrix depends upon themolecular weight of the immunoconjugate or antibody, the amount ofimmunoconjugate or antibody within the matrix, and the size of dispersedparticles. Saltzman et al., Biophys. J. 55: 163 (1989); Sherwood et al.,supra. Other solid dosage forms are described in Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea& Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

More generally, the dosage of an administered immunoconjugate for humanswill vary depending upon such factors as the patient's age, weight,height, sex, general medical condition and previous medical history. Itmay be desirable to provide the recipient with a dosage ofimmunoconjugate, antibody fusion protein that is in the range of fromabout 1 mg/kg to 25 mg/kg as a single intravenous infusion, although alower or higher dosage also may be administered as circumstancesdictate. A dosage of 1-20 mg/kg for a 70 kg patient, for example, is70-1,400 mg, or 41-824 mg/m² for a 1.7-m patient. The dosage may berepeated as needed, for example, once per week for 4-10 weeks, once perweek for 8 weeks, or once per week for 4 weeks. It may also be givenless frequently, such as every other week for several months, or monthlyor quarterly for many months, as needed in a maintenance therapy.

Alternatively, an antibody may be administered as one dosage every 2 or3 weeks, repeated for a total of at least 3 dosages. Or, the antibodiesmay be administered twice per week for 4-6 weeks. If the dosage islowered to approximately 200-300 mg/m² (340 mg per dosage for a 1.7-mpatient, or 4.9 mg/kg for a 70 kg patient), it may be administered onceor even twice weekly for 4 to 10 weeks. Alternatively, the dosageschedule may be decreased, namely every 2 or 3 weeks for 2-3 months. Ithas been determined, however, that even higher doses, such as 20 mg/kgonce weekly or once every 2-3 weeks can be administered by slow i.v.infusion, for repeated dosing cycles. The dosing schedule can optionallybe repeated at other intervals and dosage may be given through variousparenteral routes, with appropriate adjustment of the dose and schedule.

Immunoconjugates

Anti-pancreatic cancer antibodies and fragments thereof may beconjugated to at least one therapeutic and/or diagnostic agent fortherapy or diagnosis. For immunotherapy, the objective is to delivercytotoxic doses of radioactivity, toxin, antibody and/or drug to targetcells, while minimizing exposure to non-target tissues. Preferably,anti-pancreatic cancer antibodies are be used to diagnose and/or treatpancreatic tumors.

Any of the antibodies, antibody fragments and fusion proteins can beconjugated with one or more therapeutic or diagnostic agents, using avariety of techniques known in the art. One or more therapeutic ordiagnostic agents may be attached to each antibody, antibody fragment orfusion protein, for example by conjugating an agent to a carbohydratemoiety in the Fc region of the antibody. If the Fc region is absent (forexample with certain antibody fragments), it is possible to introduce acarbohydrate moiety into the light chain variable region of either anantibody or antibody fragment to which a therapeutic or diagnostic agentmay be attached. See, for example, Leung et al., J. Immunol. 154: 5919(1995); Hansen et al., U.S. Pat. No. 5,443,953 (1995), Leung et al.,U.S. Pat. No. 6,254,868, the Examples section of each patentincorporated herein by reference.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int J Cancer 41: 832 (1988); Shih et al., IntJ Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No. 5,057,313, theExamples section of which is incorporated herein by reference. Thegeneral method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function and that is loaded with a plurality oftherapeutic agents, such as peptides or drugs. This reaction results inan initial Schiff base (imine) linkage, which can be stabilized byreduction to a secondary amine to form the final conjugate.

Antibody fusion proteins or multispecific antibodies comprise two ormore antibodies or fragments thereof, each of which may be attached toat least one therapeutic agent and/or diagnostic agent. Accordingly, oneor more of the antibodies or fragments thereof of the antibody fusionprotein can have more than one therapeutic and/or diagnostic agentattached. Further, the therapeutic agents do not need to be the same butcan be different therapeutic agents, for example, one can attach a drugand a radioisotope to the same fusion protein. For example, an IgG canbe radiolabeled with ¹³¹I and attached to a drug. The ¹³¹I can beincorporated into the tyrosine of the IgG and the drug attached to theepsilon amino group of the IgG lysines. Both therapeutic and diagnosticagents also can be attached to reduced SH groups and to the carbohydrateside chains of antibodies. Alternatively, a bispecific antibody maycomprise one antibody or fragment thereof against a disease antigen andanother against a hapten attached to a targetable construct, for use inpretargeting techniques as discussed above.

A therapeutic or diagnostic agent can be attached at the hinge region ofa reduced antibody component via disulfide bond formation. As analternative, such agents can be attached to the antibody component usinga heterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLESAND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995).

Click Chemistry

An alternative method for attaching chelating moieties, drugs or otherfunctional groups to an antibody, fragment or fusion protein involvesuse of click chemistry reactions. The click chemistry approach wasoriginally conceived as a method to rapidly generate complex substancesby joining small subunits together in a modular fashion. (See, e.g.,Kolb et al., 2004, Angew Chem Int Ed 40:3004-31; Evans, 2007, Aust JChem 60:384-95.) Various forms of click chemistry reaction are known inthe art, such as the Huisgen 1,3-dipolar cycloaddition copper catalyzedreaction (Tornoe et al., 2002, J Organic Chem 67:3057-64), which isoften referred to as the “click reaction.” Other alternatives includecycloaddition reactions such as the Diels-Alder, nucleophilicsubstitution reactions (especially to small strained rings like epoxyand aziridine compounds), carbonyl chemistry formation of urea compoundsand reactions involving carbon-carbon double bonds, such as alkynes inthiol-yne reactions.

The azide alkyne Huisgen cycloaddition reaction uses a copper catalystin the presence of a reducing agent to catalyze the reaction of aterminal alkyne group attached to a first molecule. In the presence of asecond molecule comprising an azide moiety, the azide reacts with theactivated alkyne to form a 1,4-disubstituted 1,2,3-triazole. The coppercatalyzed reaction occurs at room temperature and is sufficientlyspecific that purification of the reaction product is often notrequired. (Rostovstev et al., 2002, Angew Chem Int Ed 41:2596; Tornoe etal., 2002, J Org Chem 67:3057.) The azide and alkyne functional groupsare largely inert towards biomolecules in aqueous medium, allowing thereaction to occur in complex solutions. The triazole formed ischemically stable and is not subject to enzymatic cleavage, making theclick chemistry product highly stable in biological systems. Althoughthe copper catalyst is toxic to living cells, the copper-based clickchemistry reaction may be used in vitro for immunoconjugate formation.

A copper-free click reaction has been proposed for covalent modificationof biomolecules. (See, e.g., Agard et al., 2004, J Am Chem Soc126:15046-47.) The copper-free reaction uses ring strain in place of thecopper catalyst to promote a [3+2] azide-alkyne cycloaddition reaction(Id.) For example, cyclooctyne is a 8-carbon ring structure comprisingan internal alkyne bond. The closed ring structure induces a substantialbond angle deformation of the acetylene, which is highly reactive withazide groups to form a triazole. Thus, cyclooctyne derivatives may beused for copper-free click reactions (Id.)

Another type of copper-free click reaction was reported by Ning et al.(2010, Angew Chem Int Ed 49:3065-68), involving strain-promotedalkyne-nitrone cycloaddition. To address the slow rate of the originalcyclooctyne reaction, electron-withdrawing groups are attached adjacentto the triple bond (Id.) Examples of such substituted cyclooctynesinclude difluorinated cyclooctynes, 4-dibenzocyclooctynol andazacyclooctyne (Id.) An alternative copper-free reaction involvedstrain-promoted alkyne-nitrone cycloaddition to give N-alkylatedisoxazolines (Id.) The reaction was reported to have exceptionally fastreaction kinetics and was used in a one-pot three-step protocol forsite-specific modification of peptides and proteins (Id.) Nitrones wereprepared by the condensation of appropriate aldehydes withN-methylhydroxylamine and the cycloaddition reaction took place in amixture of acetonitrile and water (Id.) These and other known clickchemistry reactions may be used to attach chelating moieties toantibodies or other targeting molecules in vitro.

Therapeutic Agents

A wide variety of therapeutic reagents can be administered concurrentlyor sequentially, or advantageously conjugated to the antibodies of theinvention, for example, drugs, toxins, oligonucleotides,immunomodulators, hormones, hormone antagonists, enzymes, enzymeinhibitors, radionuclides, angiogenesis inhibitors, pro-apoptoticagents, etc. The therapeutic agents recited here are those agents thatare useful for either conjugated to an antibody, fragment or fusionprotein or for administration separately with a naked antibody asdescribed above.

Therapeutic agents include, for example, chemotherapeutic drugs such asvinca alkaloids, anthracyclines, gemcitabine, epipodophyllotoxins,taxanes, antimetabolites, alkylating agents, antibiotics, SN-38, COX-2inhibitors, antimitotics, antiangiogenic and apoptotic agents,particularly doxorubicin, methotrexate, taxol, CPT-11, camptothecans,proteosome inhibitors, mTOR inhibitors, HDAC inhibitors, tyrosine kinaseinhibitors, and others from these and other classes of anticanceragents.

Other useful cancer chemotherapeutic drugs include nitrogen mustards,alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, COX-2inhibitors, antimetabolites, pyrimidine analogs, purine analogs,platinum coordination complexes, mTOR inhibitors, tyrosine kinaseinhibitors, proteosome inhibitors, HDAC inhibitors, camptothecins andhormones. Suitable chemotherapeutic agents are described in REMINGTON'SPHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995), and inGOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed.(MacMillan Publishing Co. 1985), as well as revised editions of thesepublications. Other suitable chemotherapeutic agents, such asexperimental drugs, are known to those of skill in the art.

In a preferred embodiment, conjugates of camptothecins and relatedcompounds, such as SN-38, may be conjugated to hPAM4 or otheranti-pancreatic cancer antibodies, for example as disclosed in U.S. Pat.No. 7,591,994; and U.S. patent application Ser. No. 11/388,032, filedMar. 23, 2006, the Examples section of each of which is incorporatedherein by reference.

In another preferred embodiment, an hPAM4 antibody is conjugated togemcitabine, which may be given before, after, or concurrently with anaked or conjugated chimeric, humanized or human PAM4 antibody.Preferably, the conjugated hPAM4 antibody or antibody fragment isconjugated to a radionuclide.

A toxin can be of animal, plant or microbial origin. A toxin, such asPseudomonas exotoxin, may also be complexed to or form the therapeuticagent portion of an immunoconjugate of the anti-pancreatic cancer andhPAM4 antibodies. Other toxins suitably employed in the preparation ofsuch conjugates or other fusion proteins, include ricin, abrin,ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, andPseudomonas endotoxin. See, for example, Pastan et al., Cell 47:641(1986), Goldenberg, CA—A Cancer Journal for Clinicians 44:43 (1994),Sharkey and Goldenberg, CA—A Cancer Journal for Clinicians 56:226(2006). Additional toxins suitable for use are known to those of skillin the art and are disclosed in U.S. Pat. No. 6,077,499, the Examplessection of which is incorporated herein by reference.

An immunomodulator, such as a cytokine, may also be conjugated to, orform the therapeutic agent portion of the immunoconjugate, or may beadministered with, but unconjugated to, an antibody, antibody fragmentor fusion protein. The fusion protein may comprise one or moreantibodies or fragments thereof binding to different antigens. Forexample, the fusion protein may bind the PAM4 antigen as well as toimmunomodulating cells or factors. Alternatively, subjects can receive anaked antibody, antibody fragment or fusion protein and a separatelyadministered cytokine, which can be administered before, concurrently orafter administration of the naked antibodies. As used herein, the term“immunomodulator” includes a cytokine, a lymphokine, a monokine, a stemcell growth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), parathyroid hormone,thyroxine, insulin, proinsulin, relaxin, prorelaxin, folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH),luteinizing hormone (LH), hepatic growth factor, prostaglandin,fibroblast growth factor, prolactin, placental lactogen, OB protein, atransforming growth factor (TGF), TGF-α, TGF-β, insulin-like growthfactor (IGF), erythropoietin, thrombopoietin, tumor necrosis factor(TNF), TNF-α, TNF-β, a mullerian-inhibiting substance, mousegonadotropin-associated peptide, inhibin, activin, vascular endothelialgrowth factor, integrin, interleukin (IL), granulocyte-colonystimulating factor (G-CSF), granulocyte macrophage-colony stimulatingfactor (GM-CSF), interferon-α, interferon-β, interferon-γ, S1 factor,IL-1, IL-1cc, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18 IL-21 and IL-25,LIF, kit-ligand, FLT-3, angiostatin, thrombospondin, endostatin and LT.

The therapeutic agent may comprise one or more radioactive isotopesuseful for treating diseased tissue. Particularly useful therapeuticradionuclides include, but are not limited to ¹¹¹In, ¹⁷⁷Lu, ²¹²Bi,²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb,²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm,¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, and ²¹¹. The therapeutic radionuclidepreferably has a decay energy in the range of 20 to 6,000 keV,preferably in the ranges 60 to 200 keV for an Auger emitter, 100-2,500keV for a beta emitter, and 4,000-6,000 keV for an alpha emitter.Maximum decay energies of useful beta-particle-emitting nuclides arepreferably 20-5,000 keV, more preferably 100-4,000 keV, and mostpreferably 500-2,500 keV. Also preferred are radionuclides thatsubstantially decay with Auger-emitting particles. For example, Co-58,Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161,Os-189m and Ir-192. Decay energies of useful beta-particle-emittingnuclides are preferably <1,000 keV, more preferably <100 keV, and mostpreferably <70 keV. Also preferred are radionuclides that substantiallydecay with generation of alpha-particles. Such radionuclides include,but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215,Bi-211, Ac-225, Fr-221, At-217, Bi-213 and Fm-255. Decay energies ofuseful alpha-particle-emitting radionuclides are preferably 2,000-10,000keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000keV.

For example, ⁶⁷Cu, considered one of the more promising radioisotopesfor radioimmunotherapy due to its 61.5-hour half-life and abundantsupply of beta particles and gamma rays, can be conjugated to anantibody using the chelating agent,p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid (TETA).Alternatively, ⁹⁰Y, which emits an energetic beta particle, can becoupled to an antibody, antibody fragment or fusion protein, usingdiethylenetriaminepentaacetic acid (DTPA), or more preferably usingDOTA. Methods of conjugating ⁹⁰Y to antibodies or targetable constructsare known in the art and any such known methods may be used. (See, e.g.,U.S. Pat. No. 7,259,249, the Examples section of which is incorporatedherein by reference. See also Linden et al., Clin Cancer Res.11:5215-22, 2005; Sharkey et al., J Nucl Med. 46:620-33, 2005; Sharkeyet al., J Nucl Med. 44:2000-18, 2003.)

Additional potential therapeutic radioisotopes include ¹¹C, ¹³N, ¹⁵O,⁷⁵Br, ¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru,¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁹⁷Pt,¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵¹Cr, ⁵⁹Fe, ⁷⁵Se,²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.

In another embodiment, a radiosensitizer can be used in combination witha naked or conjugated antibody or antibody fragment. For example, theradiosensitizer can be used in combination with a radiolabeled antibodyor antibody fragment. The addition of the radiosensitizer can result inenhanced efficacy when compared to treatment with the radiolabeledantibody or antibody fragment alone. Radiosensitizers are described inD. M. Goldenberg (ed.), CANCER THERAPY WITH RADIOLABELED ANTIBODIES, CRCPress (1995). Other typical radionsensitizers of interest for use withthis technology include gemcitabine, 5-fluorouracil, and cisplatin, andhave been used in combination with external irradiation in the therapyof diverse cancers, including pancreatic cancer. Therefore, we havestudied the combination of gemcitabine at what is believed to beradiosensitizing doses (once weekly 200 mg/m² over 4 weeks) ofgemcitabine combined with fractionated doses of ⁹⁰Y-hPAM4, and haveobserved objective evidence of pancreatic cancer reduction after asingle cycle of this combination that proved to be well-tolerated (nograde 3-4 toxicities by NCl-CTC v. 3 standard).

Antibodies or fragments thereof that have a boron addend-loaded carrierfor thermal neutron activation therapy will normally be effected insimilar ways. However, it will be advantageous to wait untilnon-targeted immunoconjugate clears before neutron irradiation isperformed. Clearance can be accelerated using an anti-idiotypic antibodythat binds to the anti-pancreatic cancer antibody. See U.S. Pat. No.4,624,846 for a description of this general principle. For example,boron addends such as carboranes, can be attached to antibodies.Carboranes can be prepared with carboxyl functions on pendant sidechains, as is well-known in the art. Attachment of carboranes to acarrier, such as aminodextran, can be achieved by activation of thecarboxyl groups of the carboranes and condensation with amines on thecarrier. The intermediate conjugate is then conjugated to the antibody.After administration of the antibody conjugate, a boron addend isactivated by thermal neutron irradiation and converted to radioactiveatoms which decay by alpha-emission to produce highly toxic, short-rangeeffects.

Interference RNA

Another type of therapeutic agent is RNAi or siRNA. RNA interference(RNAi) is mediated by the RNA-induced silencing complex (RISC) and isinitiated by short double-stranded RNA molecules that interact with thecatalytic RISC component argonaute (Rand et al., 2005, Cell 123:621-29).Types of RNAi molecules include microRNA (miRNA) and small interferingRNA (siRNA). RNAi species can bind with messenger RNA (mRNA) throughcomplementary base-pairing and inhibits gene expression bypost-transcriptional gene silencing. Upon binding to a complementarymRNA species, RNAi induces cleavage of the mRNA molecule by theargonaute component of RISC. Among other characteristics, miRNA andsiRNA differ in the degree of specificity for particular gene targets,with siRNA being relatively specific for a particular target gene andmiRNA inhibiting translation of multiple mRNA species.

Therapeutic use of RNAi by inhibition of selected gene expression hasbeen attempted for a variety of disease states, such as maculardegeneration and respiratory syncytial virus infection (Sah, 2006, LifeSci 79:1773-80). It has been suggested that siRNA functions in host celldefenses against viral infection and siRNA has been widely examined asan approach to anti-viral therapy (see, e.g., Zhang et al., 2004, NatureMed 11:56-62; Novina et al., 2002, Nature Med 8:681-86; Palliser et al.,2006, Nature 439:89-94). The use of siRNA for cancer therapy has alsobeen attempted. Fujii et al. (2006, Int J Oncol 29:541-48) transfectedHPV positive cervical cancer cells with siRNA against HPV E6 and E7 andsuppressed tumor growth. siRNA-mediated knockdown of metadherinexpression in breast cancer cells was reported to inhibit experimentallung metastasis (Brown and Ruoslahti, 2004, Cancer Cell 5:365-74).

Attempts have been made to provide targeted delivery of siRNA to reducethe potential for off-target toxicity. Song et al. (2005, Nat Biotechnol23:709-17) used protamine-conjugated Fab fragments against HIV envelopeprotein to deliver siRNA to circulating cells. Schiffelers et al. (2004,Nucl Acids Res 32:e149) conjugated RGD peptides to nanoparticles todeliver anti-VEGFR2 siRNA to tumors and inhibited tumor angiogenesis andgrowth rate in nude mice. Dickerson et al. used nanogels functionalizedwith anti-EphA2 receptor peptides to chemosensitize ovarian cancer cellswith siRNA against EGFR. Dendrimer-conjugated magnetic nanoparticleshave been applied to the targeted delivery of antisense survivinoligodeoxynucleotides (Pan et al., 2007, Cancer Res 67:8156-63).

The skilled artisan will realize that any siRNA or interference RNAspecies may be attached to the subject antibodies. siRNA and RNAispecies against a wide variety of targets are known in the art, and anysuch known oligonucleotide species may be utilized in the claimedmethods and compositions.

Known siRNA species of potential use include those specific forIKK-gamma (U.S. Pat. No. 7,022,828); VEGF, Flt-1 and Flk-1/KDR (U.S.Pat. No. 7,148,342); Bc12 and EGFR (U.S. Pat. No. 7,541,453); CDC20(U.S. Pat. No. 7,550,572); transducin (beta)-like 3 (U.S. Pat. No.7,576,196); KRAS (U.S. Pat. No. 7,576,197); carbonic anhydrase II (U.S.Pat. No. 7,579,457); complement component 3 (U.S. Pat. No. 7,582,746);interleukin-1 receptor-associated kinase 4 (IRAK4) (U.S. Pat. No.7,592,443); survivin (U.S. Pat. No. 7,608,7070); superoxide dismutase 1(U.S. Pat. No. 7,632,938); MET proto-oncogene (U.S. Pat. No. 7,632,939);amyloid beta precursor protein (APP) (U.S. Pat. No. 7,635,771); IGF-1R(U.S. Pat. No. 7,638,621); ICAM1 (U.S. Pat. No. 7,642,349); complementfactor B (U.S. Pat. No. 7,696,344); p53 (7,781,575), and apolipoproteinB (7,795,421), the Examples section of each of which is incorporatedherein by reference.

Additional siRNA species are available from known commercial sources,such as Sigma-Aldrich (St Louis, Mo.), Invitrogen (Carlsbad, Calif.),Santa Cruz Biotechnology (Santa Cruz, Calif.), Ambion (Austin, Tex.),Dharmacon (Thermo Scientific, Lafayette, Colo.), Promega (Madison,Wis.), Minis Bio (Madison, Wis.) and Qiagen (Valencia, Calif.), amongmany others. Other publicly available sources of siRNA species includethe siRNAdb database at the Stockholm Bioinformatics Centre, theMIT/ICBP siRNA Database, the RNAi Consortium shRNA Library at the BroadInstitute, and the Probe database at NCBI. For example, there are 30,852siRNA species in the NCBI Probe database. The skilled artisan willrealize that for any gene of interest, either an siRNA species hasalready been designed, or one may readily be designed using publiclyavailable software tools. Any such siRNA species may be delivered usingthe subject DNL complexes.

Exemplary siRNA species that have been reported are listed in Table 1.Although siRNA is delivered as a double-stranded molecule, forsimplicity only the sense strand sequences are shown in Table 1.

TABLE 1 Exemplary siRNA Sequences Target Sequence SEQ ID NO VEGF R2AATGCGGCGGTGGTGACAGTA SEQ ID NO: 22 VEGF R2 AAGCTCAGCACACAGAAAGACSEQ ID NO: 23 CXCR4 UAAAAUCUUCCUGCCCACCdTdT SEQ ID NO: 24 CXCR4GGAAGCUGUUGGCUGAAAAdTdT SEQ ID NO: 25 PPARC1 AAGACCAGCCUCUUUGCCCAGSEQ ID NO: 26 Dynamin 2 GGACCAGGCAGAAAACGAG SEQ ID NO: 27 CateninCUAUCAGGAUGACGCGG SEQ ID NO: 29 E1A binding  UGACACAGGCAGGCUUGACUUSEQ ID NO: 29 protein Plasminogen GGTGAAGAAGGGCGTCCAA SEQ ID NO: 30activator K-ras GATCCGTTGGAGCTGTTGGCGTAGTT SEQ ID NO: 31CAAGAGACTCGCCAACAGCTCCAACT TTTGGAAA Sortilin 1 AGGTGGTGTTAACAGCAGAGSEQ ID NO: 32 Apolipoprotein E AAGGTGGAGCAAGCGGTGGAG SEQ ID NO: 33Apolipoprotein E AAGGAGTTGAAGGCCGACAAA SEQ ID NO: 34 Bcl-XUAUGGAGCUGCAGAGGAUGdTdT SEQ ID NO: 35 Raf-1 TTTGAATATCTGTGCTGAGAACACASEQ ID NO: 36 GTTCTCAGCACAGATATTCTTTTT Heat shockAATGAGAAAAGCAAAAGGTGCCCTGTCTC SEQ ID NO: 37 transcription  factor 2IGFBP3 AAUCAUCAUCAAGAAAGGGCA SEQ ID NO: 38 ThioredoxinAUGACUGUCAGGAUGUUGCdTdT SEQ ID NO: 39 CD44 GAACGAAUCCUGAAGACAUCUSEQ ID NO: 40 MMP14 AAGCCTGGCTACAGCAATATGCCTGTCTC SEQ ID NO: 41 MAPKAPK2UGACCAUCACCGAGUUUAUdTdT SEQ ID NO: 42 FGFR1 AAGTCGGACGCAACAGAGAAASEQ ID NO: 43 ERBB2 CUACCUUUCUACGGACGUGdTdT SEQ ID NO: 44 BCL2L1CTGCCTAAGGCGGATTTGAAT SEQ ID NO: 45 ABL1 TTAUUCCUUCUUCGGGAAGUCSEQ ID NO: 46 CEACAM1 AACCTTCTGGAACCCGCCCAC SEQ ID NO: 47 CD9GAGCATCTTCGAGCAAGAA SEQ ID NO: 48 CD151 CATGTGGCACCGTTTGCCTSEQ ID NO: 49 Caspase 8 AACTACCAGAAAGGTATACCT SEQ ID NO: 50 BRCA1UCACAGUGUCCUUUAUGUAdTdT SEQ ID NO: 51 p53 GCAUGAACCGGAGGCCCAUTTSEQ ID NO: 52 CEACAM6 CCGGACAGTTCCATGTATA SEQ ID NO: 53

Diagnostic Agents

In the context of this application, the terms “diagnosis” or “detection”can be used interchangeably. Whereas diagnosis usually refers todefining a tissue's specific histological status, detection recognizesand locates a tissue, lesion or organism containing a particularantigen.

The subject antibodies and fragments can be detectably labeled bylinking the antibody to an enzyme. When the antibody-enzyme conjugate isincubated in the presence of the appropriate substrate, the enzymemoiety reacts with the substrate to produce a chemical moiety which canbe detected, for example, by spectrophotometric, fluorometric or visualmeans. Examples of enzymes that can be used to detectably label antibodyinclude malate dehydrogenase, staphylococcal nuclease, delta-V-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphatedehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase,alpha-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphatedehydrogenase, glucoamylase and acetylcholinesterase.

The immunoconjugate may comprise one or more radioactive isotopes usefulfor detecting diseased tissue. Particularly useful diagnosticradionuclides include, but are not limited to, ¹¹⁰In, ¹¹¹In, ¹⁷⁷Lu, ¹⁸F,⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr, ^(94m)Tc, ⁹⁴Tc,^(99m)Tc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ³²P, ¹¹C, ¹³N, ¹⁵O,¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ^(52m)Mn, ⁵⁵Co, ⁷²As, ⁷⁵Br, ⁷⁶Br, ^(82m)Rb, ⁸³Sr, orother gamma-, beta-, or positron-emitters, preferably with a decayenergy in the range of 20 to 4,000 keV, more preferably in the range of25 to 4,000 keV, and even more preferably in the range of 25 to 1,000keV, and still more preferably in the range of 70 to 700 keV. Totaldecay energies of useful positron-emitting radionuclides are preferably<2,000 keV, more preferably under 1,000 keV, and most preferably <700keV. Radionuclides useful as diagnostic agents utilizing gamma-raydetection include, but are not limited to: ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶⁷Cu,⁶⁷Ga, ⁷⁵Se, ⁹⁷Ru, ^(99m)Tc, ¹¹¹In, ^(114m)In, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁶⁹Yb,¹⁹⁷Hg, and ²⁰¹Tl. Decay energies of useful gamma-ray emittingradionuclides are preferably 20-2000 keV, more preferably 60-600 keV,and most preferably 100-300 keV.

Methods of diagnosing cancer in a subject may be accomplished byadministering a diagnostic immunoconjugate and detecting the diagnosticlabel attached to an immunoconjugate that is localized to a cancer ortumor. The antibodies, antibody fragments and fusion proteins may beconjugated to the diagnostic agent or may be administered in apretargeting technique using targetable constructs attached to adiagnostic agent. Radioactive agents that can be used as diagnosticagents are discussed above. A suitable non-radioactive diagnostic agentis a contrast agent suitable for magnetic resonance imaging, X-rays,computed tomography or ultrasound. Magnetic imaging agents include, forexample, non-radioactive metals, such as manganese, iron and gadolinium,complexed with metal-chelate combinations that include 2-benzyl-DTPA andits monomethyl and cyclohexyl analogs. See U.S. Ser. No. 09/921,290filed on Oct. 10, 2001, the Examples section of which is incorporatedherein by reference. Other imaging agents such as PET scanningnucleotides, preferably ¹⁸F, may also be used.

Contrast agents, such as MRI contrast agents, including, for example,gadolinium ions, lanthanum ions, dysprosium ions, iron ions, manganeseions or other comparable labels, CT contrast agents, and ultrasoundcontrast agents may be used as diagnostic agents. Paramagnetic ionssuitable for use include chromium (III), manganese (II), iron (III),iron (II), cobalt (II), nickel (II), copper (II), neodymium (III),samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) and erbium (III), withgadolinium being particularly preferred.

Ions useful in other contexts, such as X-ray imaging, include but arenot limited to lanthanum (III), gold (III), lead (II) and bismuth (III).Fluorescent labels include rhodamine, fluorescein and renographin.Rhodamine and fluorescein are often linked via an isothiocyanateintermediate.

Metals are also useful in diagnostic agents, including those formagnetic resonance imaging techniques. These metals include, but are notlimited to: gadolinium, manganese, iron, chromium, copper, cobalt,nickel, dysprosium, rhenium, europium, terbium, holmium and neodymium.In order to load an antibody with radioactive metals or paramagneticions, it may be necessary to react it with a reagent having a long tailto which are attached a multiplicity of chelating groups for binding theions. Such a tail can be a polymer such as a polylysine, polysaccharide,or other derivatized or derivatizable chain having pendant groups towhich can be bound chelating groups such as, e.g.,ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), porphyrins, polyamines, crown ethers,bis-thiosemicarbazones, polyoximes, and like groups known to be usefulfor this purpose.

Chelates are coupled to an antibody, fusion protein, or fragmentsthereof using standard chemistries. The chelate is normally linked tothe antibody by a group which enables formation of a bond to themolecule with minimal loss of immunoreactivity and minimal aggregationand/or internal cross-linking. Other, more unusual, methods and reagentsfor conjugating chelates to antibodies are disclosed in U.S. Pat. No.4,824,659 to Hawthorne, entitled “Antibody Conjugates”, issued Apr. 25,1989, the Examples section of which is incorporated herein by reference.Particularly useful metal-chelate combinations include 2-benzyl-DTPA andits monomethyl and cyclohexyl analogs, used with diagnostic isotopes inthe general energy range of 20 to 2,000 keV. The same chelates, whencomplexed with non-radioactive metals, such as manganese, iron andgadolinium are useful for MRI. Macrocyclic chelates such as NOTA, DOTA,and TETA are of use with a variety of metals and radiometals, mostparticularly with radionuclides of gallium, yttrium and copper,respectively. Such metal-chelate complexes can be made very stable bytailoring the ring size to the metal of interest. Other ring-typechelates such as macrocyclic polyethers, which are of interest forstably binding nuclides, such as 223Ra for RAIT are encompassed by theinvention.

Radiopaque and contrast materials are used for enhancing X-rays andcomputed tomography, and include iodine compounds, barium compounds,gallium compounds, thallium compounds, etc. Specific compounds includebarium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid,iocetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide,iohexyl, iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid,ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, iotetricacid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid,ipodate, meglumine, metrizamide, metrizoate, propyliodone, and thallouschloride.

The antibodies, antibody fragments and fusion proteins also can belabeled with a fluorescent compound. The presence of afluorescent-labeled MAb is determined by exposing the antibody to lightof the proper wavelength and detecting the resultant fluorescence.Fluorescent labeling compounds include Alexa 350, Alexa 430, AMCA,aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G,BODIPY-TMR, BODIPY-TRX, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein,5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine,6-carboxytetramethyl amino, Cascade Blue, Cy2, Cy3, Cy5,6-FAM, dansylchloride, Fluorescein, fluorescein isothiocyanate, fluorescamine, HEX,6-JOE, NBD (7-nitrobenz-2-oxa-1,3-diazole), Oregon Green 488, OregonGreen 500, Oregon Green 514, Pacific Blue, phthalic acid, terephthalicacid, isophthalic acid, cresyl fast violet, cresyl blue violet,brilliant cresyl blue, para-aminobenzoic acid, erythrosine,phthalocyanines, phthaldehyde, azomethines, cyanines, xanthines,succinylfluoresceins, rare earth metal cryptates, europiumtrisbipyridine diamine, a europium cryptate or chelate, diamine,dicyanins, La Jolla blue dye, allopycocyanin, allococyanin B,phycocyanin C, phycocyanin R, thiamine, phycoerythrocyanin,phycoerythrin R, REG, Rhodamine Green, rhodamine isothiocyanate,Rhodamine Red, ROX, TAMRA, TET, TRIT (tetramethyl rhodamine isothiol),Tetramethylrhodamine, and Texas Red. Fluorescently-labeled antibodiesare particularly useful for flow cytometry analysis, but can also beused in endoscopic and intravascular detection methods.

Alternatively, the antibodies, antibody fragments and fusion proteinscan be detectably labeled by coupling the antibody to a chemiluminescentcompound. The presence of the chemiluminescent-tagged MAb is determinedby detecting the presence of luminescence that arises during the courseof a chemical reaction. Examples of chemiluminescent labeling compoundsinclude luminol, isoluminol, an aromatic acridinium ester, an imidazole,an acridinium salt and an oxalate ester.

Similarly, a bioluminescent compound can be used to label the antibodiesand fragments there. Bioluminescence is a type of chemiluminescencefound in biological systems in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Bioluminescent compounds that are useful for labelinginclude luciferin, luciferase and aequorin.

Accordingly, a method of diagnosing a malignancy in a subject isdescribed, comprising performing an in vitro diagnosis assay on aspecimen (fluid, tissue or cells) from the subject with a compositioncomprising an anti-pancreatic cancer MAb, fusion protein or fragmentthereof. Immunohistochemistry can be used to detect the presence of PAM4antigen in a cell or tissue by the presence of bound antibody.Preferably, the malignancy that is being diagnosed is a cancer. Mostpreferably, the cancer is pancreatic cancer.

Additionally, a chelator such as DTPA, DOTA, TETA, or NOTA or a suitablepeptide, to which a detectable label, such as a fluorescent molecule, orcytotoxic agent, such as a heavy metal or radionuclide, can beconjugated to a subject antibody. For example, a therapeutically usefulimmunoconjugate can be obtained by conjugating a photoactive agent ordye to an antibody fusion protein. Fluorescent compositions, such asfluorochrome, and other chromogens, or dyes, such as porphyrinssensitive to visible light, have been used to detect and to treatlesions by directing the suitable light to the lesion. In therapy, thishas been termed photoradiation, phototherapy, or photodynamic therapy(Jori et al. (eds.), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES(Libreria Progetto 1985); van den Bergh, Chem. Britain 22:430 (1986)).Moreover, monoclonal antibodies have been coupled with photoactivateddyes for achieving phototherapy. Mew et al., J. Immunol. 130:1473(1983); idem., Cancer Res. 45:4380 (1985); Oseroff et al., Proc Natl.Acad. Sci. USA 83:8744 (1986); idem., Photochem. Photobiol. 46:83(1987); Hasan et al., Prog. Clin. Biol. Res. 288:471 (1989); Tatsuta etal., Lasers Surg. Med. 9:422 (1989); Pelegrin et al., Cancer 67:2529(1991).

Fluorescent and radioactive agents conjugated to antibodies or used inbispecific, pretargeting methods, are particularly useful forendoscopic, intraoperative or intravascular detection of the targetedantigens associated with diseased tissues or clusters of cells, such asmalignant tumors, as disclosed in Goldenberg U.S. Pat. Nos. 5,716,595;6,096,289 and 6,387,350, the Examples section of each incorporatedherein by reference, particularly with gamma-, beta- andpositron-emitters. Endoscopic applications may be used when there isspread to a structure that allows an endoscope, such as the colon.Radionuclides useful for positron emission tomography include, but arenot limited to: F-18, Mn-51, Mn-52m, Fe-52, Co-55, Cu-62, Cu-64, Ga-68,As-72, Br-75, Br-76, Rb-82m, Sr-83, Y-86, Zr-89, Tc-94m, In-110, 1-120,and 1-124. Total decay energies of useful positron-emittingradionuclides are preferably <2,000 keV, more preferably under 1,000keV, and most preferably <700 keV. Radionuclides useful as diagnosticagents utilizing gamma-ray detection include, but are not limited to:Cr-51, Co-57, Co-58, Fe-59, Cu-67, Ga-67, Se-75, R^(u)-97, Tc-99m,In-111, In-114m, I-123, I-125, I-131, Yb-169, Hg-197, and T1-201. Decayenergies of useful gamma-ray emitting radionuclides are preferably20-2000 keV, more preferably 60-600 keV, and most preferably 100-300keV.

In Vitro Diagnosis

The present invention contemplates the use of anti-pancreatic cancerantibodies to screen biological samples in vitro for the presence of thePAM4 antigen. In such immunoassays, the antibody, antibody fragment orfusion protein may be utilized in liquid phase or bound to a solid-phasecarrier, as described below. For purposes of in vitro diagnosis, anytype of antibody such as murine, chimeric, humanized or human may beutilized, since there is no host immune response to consider.

One example of a screening method for determining whether a biologicalsample contains the PAM4 antigen is the radioimmunoassay (RIA). Forexample, in one form of RIA, the substance under test is mixed with PAM4MAb in the presence of radiolabeled PAM4 antigen. In this method, theconcentration of the test substance will be inversely proportional tothe amount of labeled PAM4 antigen bound to the MAb and directly relatedto the amount of free, labeled PAM4 antigen. Other suitable screeningmethods will be readily apparent to those of skill in the art.

Alternatively, in vitro assays can be performed in which ananti-pancreatic cancer antibody, antibody fragment or fusion protein isbound to a solid-phase carrier. For example, MAbs can be attached to apolymer, such as aminodextran, in order to link the MAb to an insolublesupport such as a polymer-coated bead, a plate or a tube.

Other suitable in vitro assays will be readily apparent to those ofskill in the art. The specific concentrations of detectably labeledantibody and PAM4 antigen, the temperature and time of incubation, aswell as other assay conditions may be varied, depending on variousfactors including the concentration of the PAM4 antigen in the sample,the nature of the sample, and the like. The binding activity of a sampleof anti-pancreatic cancer antibody may be determined according towell-known methods. Those skilled in the art will be able to determineoperative and optimal assay conditions for each determination byemploying routine experimentation.

The presence of the PAM4 antigen in a biological sample can bedetermined using an enzyme-linked immunosorbent assay (ELISA) (e.g.,Gold et al. J Clin Oncol. 24:252-58, 2006). In the direct competitiveELISA, a pure or semipure antigen preparation is bound to a solidsupport that is insoluble in the fluid or cellular extract being testedand a quantity of detectably labeled soluble antibody is added to permitdetection and/or quantitation of the binary complex formed betweensolid-phase antigen and labeled antibody.

In contrast, a “double-determinant” ELISA, also known as a “two-siteELISA” or “sandwich assay,” requires small amounts of antigen and theassay does not require extensive purification of the antigen. Thus, thedouble-determinant ELISA is preferred to the direct competitive ELISAfor the detection of an antigen in a clinical sample. See, for example,the use of the double-determinant ELISA for quantitation of the c-myconcoprotein in biopsy specimens. Field et al., Oncogene 4: 1463 (1989);Spandidos et al., AntiCancer Res. 9: 821 (1989).

In a double-determinant ELISA, a quantity of unlabeled MAb or antibodyfragment (the “capture antibody”) is bound to a solid support, the testsample is brought into contact with the capture antibody, and a quantityof detectably labeled soluble antibody (or antibody fragment) is addedto permit detection and/or quantitation of the ternary complex formedbetween the capture antibody, antigen, and labeled antibody. In thepresent context, an antibody fragment is a portion of an anti-pancreaticcancer MAb that binds to an epitope of the PAM4 antigen. Methods ofperforming a double-determinant ELISA are well-known. See, for example,Field et al., supra, Spandidos et al., supra, and Moore et al.,“Twin-Site ELISAs for fos and myc Oncoproteins Using the AMPAK System,”in METHODS 1N MOLECULAR BIOLOGY, VOL. 10, pages 273-281 (The HumanaPress, Inc. 1992). In the double-determinant ELISA, the soluble antibodyor antibody fragment must bind to a PAM4 epitope that is distinct fromthe epitope recognized by the capture antibody. The double-determinantELISA can be performed to ascertain whether the PAM4 antigen is presentin a biopsy sample. Alternatively, the assay can be performed toquantitate the amount of PAM4 antigen that is present in a clinicalsample of body fluid. The quantitative assay can be performed byincluding dilutions of purified PAM4 antigen.

The anti-pancreatic cancer MAbs, fusion proteins, and fragments thereofalso are suited for the preparation of an assay kit. Such a kit maycomprise a carrier means that is compartmentalized to receive in closeconfinement one or more container means such as vials, tubes and thelike, each of said container means comprising the separate elements ofthe immunoassay.

The subject antibodies, antibody fragments and fusion proteins also canbe used to detect the presence of the PAM4 antigen in tissue sectionsprepared from a histological specimen. Such in situ detection can beused to determine the presence of the PAM4 antigen and to determine thedistribution of the PAM4 antigen in the examined tissue. In situdetection can be accomplished by applying a detectably-labeled antibodyto frozen tissue sections. Studies indicate that the PAM4 antigen ispreserved in paraffin-embedded sections. General techniques of in situdetection are well-known to those of ordinary skill. See, for example,Ponder, “Cell Marking Techniques and Their Application,” in MAMMALIANDEVELOPMENT: A PRACTICAL APPROACH 113-38 Monk (ed.) (IRL Press 1987),and Coligan at pages 5.8.1-5.8.8.

Antibodies, antibody fragments and fusion proteins can be detectablylabeled with any appropriate marker moiety, for example, a radioisotope,an enzyme, a fluorescent label, a dye, a chromogen, a chemiluminescentlabel, a bioluminescent labels or a paramagnetic label.

The marker moiety can be a radioisotope that is detected by such meansas the use of a gamma counter or a scintillation counter or byautoradiography. In a preferred embodiment, the diagnostic conjugate isa gamma-, beta- or a positron-emitting isotope. A marker moiety in thepresent description refers to a molecule that will generate a signalunder predetermined conditions. Examples of marker moieties includeradioisotopes, enzymes, fluorescent labels, chemiluminescent labels,bioluminescent labels and paramagnetic labels.

The binding of marker moieties to anti-pancreatic cancer antibodies canbe accomplished using standard techniques known to the art. Typicalmethodology in this regard is described by Kennedy et al., Clin ChimActa 70:1 (1976), Schurs et al., Clin. Chim. Acta 81:1 (1977), Shih etal., Int J Cancer 46: 1101 (1990).

The above-described in vitro and in situ detection methods may be usedto assist in the diagnosis or staging of a pathological condition. Forexample, such methods can be used to detect tumors that express the PAM4antigen such as pancreatic cancer.

In Vivo Diagnosis/Detection

Various methods of in vivo diagnostic imaging with radiolabeled MAbs arewell-known. In the technique of immunoscintigraphy, for example,antibodies are labeled with a gamma-emitting radioisotope and introducedinto a patient. A gamma camera is used to detect the location anddistribution of gamma-emitting radioisotopes. See, for example,Srivastava (ed.), RADIOLABELED MONOCLONAL ANTIBODIES FOR IMAGING ANDTHERAPY (Plenum Press 1988), Chase, “Medical Applications ofRadioisotopes,” in REMINGTON′S PHARMACEUTICAL SCIENCES, 18th Edition,Gennaro et al. (eds.), pp. 624-652 (Mack Publishing Co., 1990), andBrown, “Clinical Use of Monoclonal Antibodies,” in BIOTECHNOLOGY ANDPHARMACY 227-49, Pezzuto et al. (eds.) (Chapman & Hall 1993).

For diagnostic imaging, radioisotopes may be bound to antibody eitherdirectly or indirectly by using an intermediary functional group. Usefulintermediary functional groups include chelators such asethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid.For example, see Shih et al., supra, and U.S. Pat. No. 5,057,313.

The radiation dose delivered to the patient is maintained at as low alevel as possible through the choice of isotope for the best combinationof minimum half-life, minimum retention in the body, and minimumquantity of isotope which will permit detection and accuratemeasurement. Examples of radioisotopes that can be bound toanti-pancreatic cancer antibody and are appropriate for diagnosticimaging include ^(99m)Tc, ¹¹¹In and ¹⁸F.

The subject antibodies, antibody fragments and fusion proteins also canbe labeled with paramagnetic ions and a variety of radiological contrastagents for purposes of in vivo diagnosis. Contrast agents that areparticularly useful for magnetic resonance imaging comprise gadolinium,manganese, dysprosium, lanthanum, or iron ions. Additional agentsinclude chromium, copper, cobalt, nickel, rhenium, europium, terbium,holmium, or neodymium. Antibodies and fragments thereof can also beconjugated to ultrasound contrast/enhancing agents. For example, oneultrasound contrast agent is a liposome. Also preferred, the ultrasoundcontrast agent is a liposome that is gas filled.

In a preferred embodiment, a bispecific antibody can be conjugated to acontrast agent. For example, the bispecific antibody may comprise morethan one image-enhancing agent for use in ultrasound imaging. In anotherpreferred embodiment, the contrast agent is a liposome. Preferably, theliposome comprises a bivalent DTPA-peptide covalently attached to theoutside surface of the liposome.

Pharmaceutically Suitable Excipients

Additional pharmaceutical methods may be employed to control theduration of action of an anti-pancreatic cancer antibody in atherapeutic application. Control release preparations can be preparedthrough the use of polymers to complex or adsorb the antibody, antibodyfragment or fusion protein. For example, biocompatible polymers includematrices of poly(ethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebacic acid.Sherwood et al., Bio/Technology 10: 1446 (1992). The rate of release ofan antibody, antibody fragment or fusion protein from such a matrixdepends upon the molecular weight of the antibody, antibody fragment orfusion protein, the amount of antibody within the matrix, and the sizeof dispersed particles. Saltzman et al., Biophys. J. 55: 163 (1989);Sherwood et al., supra. Other solid dosage forms are described in Anselet al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5thEdition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990),and revised editions thereof.

The antibodies, fragments thereof or fusion proteins to be delivered toa subject can comprise one or more pharmaceutically suitable excipients,one or more additional ingredients, or some combination of these. Theantibody can be formulated according to known methods to preparepharmaceutically useful compositions, whereby the immunoconjugate ornaked antibody is combined in a mixture with a pharmaceutically suitableexcipient. Sterile phosphate-buffered saline is one example of apharmaceutically suitable excipient. Other suitable excipients arewell-known to those in the art. See, for example, Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea& Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

The immunoconjugate or naked antibody can be formulated for intravenousadministration via, for example, bolus injection or continuous infusion.Formulations for injection can be presented in unit dosage form, e.g.,in ampules or in multi-dose containers, with an added preservative. Thecompositions can take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and can contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The immunoconjugate, naked antibody, fragment thereof or fusion proteinmay also be administered to a mammal subcutaneously or by otherparenteral routes. In a preferred embodiment, the antibody or fragmentthereof is administered in a dosage of 20 to 2000 milligrams protein perdose. Moreover, the administration may be by continuous infusion or bysingle or multiple boluses. In general, the dosage of an administeredimmunoconjugate, fusion protein or naked antibody for humans will varydepending upon such factors as the patient's age, weight, height, sex,general medical condition and previous medical history. Typically, it isdesirable to provide the recipient with a dosage of immunoconjugate,antibody fusion protein or naked antibody that is in the range of fromabout 1 mg/kg to 20 mg/kg as a single intravenous or infusion, althougha lower or higher dosage also may be administered as circumstancesdictate. This dosage may be repeated as needed, for example, once perweek for four to ten weeks, preferably once per week for eight weeks,and more preferably, once per week for four weeks. It may also be givenless frequently, such as every other week for several months, or morefrequently, such as two- or three-time weekly. The dosage may be giventhrough various parenteral routes, with appropriate adjustment of thedose and schedule.

Kits

Various embodiments may concern kits containing components suitable fortreating or diagnosing diseased tissue in a patient. Exemplary kits maycontain at least one antibody, antigen binding fragment or fusionprotein as described herein. If the composition containing componentsfor administration is not formulated for delivery via the alimentarycanal, such as by oral delivery, a device capable of delivering the kitcomponents through some other route may be included. One type of device,for applications such as parenteral delivery, is a syringe that is usedto inject the composition into the body of a subject. Inhalation devicesmay also be used. In certain embodiments, an anti-pancreatic cancerantibody or antigen binding fragment thereof may be provided in the formof a prefilled syringe or autoinjection pen containing a sterile, liquidformulation or lyophilized preparation of antibody (e.g., Kivitz et al.,Clin. Ther. 2006, 28:1619-29).

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions for use of the kit.

EXAMPLES

The examples below are illustrative of embodiments of the currentinvention and are not limiting to the scope of the claims. The examplesdiscuss studies employing an exemplary anti-pancreatic cancer monoclonalantibody (e.g., PAM4). Clinical studies with the PAM4 MAb have shownthat a majority of pancreatic cancer lesions were targeted in patientsand there was no indication of uptake in normal tissues. Dosimetryindicated that it was possible to deliver 10 to 20 cGy/mCi to tumors,with a tumor to red marrow dose ratio of 3:1 to 10:1. The data show thatPAM4 is useful for the treatment of pancreatic cancer.

Example 1 Humanized PAM4 MAb

In preferred embodiments, the claimed methods and compositions utilizethe antibody hPAM4 which is a humanized IgG of the murine PAM4 MAbraised against pancreatic cancer mucin. Humanization of the murine PAM4sequences was utilized to reduce the human antimouse antibody (HAMA)response. To produce the humanized PAM4, murine complementaritydetermining regions (CDR) were transferred from heavy and light variablechains of the mouse immunoglobulin into human framework region (FR)antibody sequences, followed by the replacement of some human FRresidues with their murine counterparts. Humanized monoclonal antibodiesare suitable for use in in vitro and in vivo diagnostic and therapeuticmethods.

Comparison of the variable region framework sequences of the murine PAM4MAb (FIGS. 1A and 1B) to known human antibodies in the Kabat databaseshowed that the FRs of PAM4 V_(k) and V_(H) exhibited the highest degreeof sequence homology to that of the human antibodies Walker V_(k) andWil2 V_(H), respectively. Therefore, the Walker V_(k) and Wi12 V_(H) FRswere selected as the human frameworks into which the murine CDRs forPAM4 V_(k) and V_(H) were grafted, respectively (FIG. 3). The FR4sequence of the human antibody, NEWM, however, was used to replace theWi12 FR4 sequence for the humanization of the PAM4 heavy chain (FIG.3B). A few amino acid residues in PAM4 FRs that flank the putative CDRswere maintained in hPAM4 based on the consideration that these residueshave more impact on Ag binding than other FR residues. These residueswere 21M, 47W, 59P, 60A, 85S, 87F, and 100G of V_(k) and 27Y, 30P, 38K,48I, 66K, 67A, and 69 L of V_(H). The DNA and amino acid sequences ofhPAM4 V_(k) (SEQ ID NO:16) and V_(H) (SEQ ID NO:19) are shown in FIGS.3A and 3B, respectively.

A modified strategy as described by Leung et al. (Leung et al., 1994))was used to construct the designed V_(k) and V_(H) genes (FIG. 4) forhPAM4 using a combination of long oligonucleotide syntheses and PCR. Forthe construction of the hPAM4 V_(H) domain, two long oligonucleotides,hPAM4 V_(H) A (173-mer) and hPAM4 V_(H) B (173-mer) were synthesized onan automated DNA synthesizer (Applied Biosystems).

hPAM4 V_(H) A represents nt 17 to 189 of the hPAM4 V_(H) domain.

(SEQ ID NO: 54) 5′-AGTCTGGGGC TGAGGTGAAG AAGCCTGGGG CCTCAGTGAA GGTCTCCTGC GAGGCTTCTG GATACACATT CCCTAGCTAT GTTTTGCACT GGGTGAAGCA GGCCCCTGGA CAAGGGCTTG AGTGGATTGG ATATATTAAT CCTTACAATG ATGGTACTCA  GTACAATGAG AAG-3′

hPAM4 V_(H)B represents the minus strand of the hPAM4 V_(H) domaincomplementary to nt 169 to 341.

(SEQ ID NO: 55) 5′-AGGGTTCCCT GGCCCCAGTA AGCAAATCCG TAGCTACCAC CGAAGCCTCT TGCACAGTAA TACACGGCCG TGTCGTCAGA TCTCAGCCTG CTCAGCTCCA TGTAGGCTGT GTTGATGGAC GTGTCCCTGG TCAGTGTGGC CTTGCCTTTG AACTTCTCAT  TGTACTGAGT ACC-3′

The 3′-terminal sequences (21 nt residues) of hPAM4 V_(H)A and V_(H)Bare complementary to each other. Under defined PCR condition, the3′-ends of hPAM4 V_(H)A and V_(H)B anneal to form a short doublestranded DNA flanked by the rest of the long oligonucleotides. Eachannealed end serves as a primer for the transcription of the singlestranded DNA, resulting in a double strand DNA composed of the nt 17 to341 of hPAM4 V_(H). This DNA was further amplified in the presence oftwo short oligonucleotides, hPAM4 V_(H)BACK and hPAM4 V_(H)FOR to formthe full-length hPAM4 V_(H). The underlined portions are restrictionsites for subcloning as shown in FIG. 4B.

hPAM4 V_(H)BACK (SEQ ID NO: 56)5′-CAG GTG CAG CTG CAG CAG TCT GGG GCT GAG GTG A-3′ hPAM4 V_(H)FOR(SEQ ID NO: 57) 5′-TGA GGA GAC GGT GAC CAG GGT TCC CTG GCC CCA-3′

A minimal amount of hPAM4 V_(H)A and V_(H)B (determined empirically) wasamplified in the presence of 10 μL of 10×PCR Buffer (500 mM KCl, 100 mMTris HCl buffer, pH 8.3, 15 mM MgCl₂), 2 μmol of hPAM4 V_(H)BACK andhPAM4 V_(k)FOR, and 2.5 units of Taq DNA polymerase (Perkin Elmer Cetus,Norwalk, Conn.). This reaction mixture was subjected to three cycles ofpolymerase chain reaction (PCR) consisting of denaturation at 94° C. for1 minute, annealing at 45° C. for 1 minute, and polymerization at 72° C.for 1.5 minutes. This procedure was followed by 27 cycles of PCRreaction consisting of denaturation at 94° C. for 1 minute, annealing at55° C. for 1 minute, and polymerization at 72° C. for 1 minute.Double-stranded PCR-amplified product for hPAM4 V_(H) was gel-purified,restriction-digested with PstI and BstEII restriction sites and clonedinto the complementary PstI/BstEII restriction sites of the heavy chainstaging vector, V_(H)pBS2, in which the V_(H) sequence was fullyassembled with the DNA sequence encoding the translation initiationcodon and a secretion signal peptide in-frame ligated at the 5′-end andan intron sequence at the 3′-end. V_(H)pBS2 is a modified staging vectorof V_(H)pBS (Leung et al., Hybridoma, 13:469, 1994), into which a XhoIrestriction site was introduced at sixteen bases upstream of thetranslation initiation codon to facilitate the next subcloning step. Theassembled V_(H) gene was subcloned as a XhoI-BamHI restriction fragmentinto the expression vector, pdHL2, which contains the expressioncassettes for both human IgG heavy and light chains under the control ofIgH enhancer and MT1 promoter, as well as a mouse d/fr gene as a markerfor selection and amplification. Since the heavy chain region of pdHL2lacks a BamHI restriction site, this ligation requires use of a linkerto provide a bridge between the BamHI site of the variable chain and theHindIII site present in the pdHL2 vector. The resulting expressionvectors were designated as hPAM4 V_(H)pdHL2.

For constructing the full length DNA of the humanized VK sequence, hPAM4V_(k)A (157-mer) and hPAM4 V_(k)B (156-mer) were synthesized asdescribed above. hPAM4 V_(k)A and V_(K)B were amplified by two shortoligonucleotides hPAM4 V_(K)BACK and hPAM4 V_(K)FOR as described above.

hPAM4 V_(K)A represents nt 16 to 172 of the hPAM4 V_(K) domain.

(SEQ ID NO: 58) 5′-CAGTCTCCAT CCTCCCTGTC TGCATCTGTA GGAGACAGAG TCACCATGAC CTGCAGTGCC AGCTCAAGTG TAAGTTCCAG CTACTTGTAC TGGTACCAAC AGAAACCAGG GAAAGCCCCC AAACTCTGGA TTTATAGCAC ATCCAACCTG GCTTCTG-3′

hPAM4 V_(K)B represents the minus strand of the hPAM4 V_(K) domaincomplementary to nt 153 to 308.

(SEQ ID NO: 59) 5′-GTCCCCCCTC CGAACGTGTA CGGGTACCTA TTCCACTGATGGCAGAAATA AGAGGCAGAA TCTTCAGGTT GCAGACTGCTGATGGTGAGA GTGAAGTCTG TCCCAGATCC ACTGCCACTGAAGCGAGCAG GGACTCCAGA AGCCAGGTTG GATGTG-3′

The 3′-terminal sequences (20 nt residues) of hPAM4 V_(K)A and V_(K)Bare complementary to each other. Under defined PCR condition, the3′-ends of hPAM4 V_(K)A and V_(K)B anneal to form a shortdouble-stranded DNA flanked by the rest of the long oligonucleotides.Each annealed end served as a primer for the transcription of the singlestranded DNA, resulting in a double strand DNA composed of nt 16 to 308of hPAM4 V_(K). This DNA was further amplified in the presence of twoshort oligonucleotides, hPAM4 V_(K)BACK and hPAM4 V_(K)FOR to form thefull-length hPAM4 V_(K). The underlined portions are restriction sitesfor subcloning as described below.

hPAM4 V_(K)BACK (SEQ ID NO: 60)5′-GAC ATC CAG CTG ACC CAG TCT CCA TCC TCC CTG-3′ hPAM4 V_(K)FOR(SEQ ID NO: 61) 5′-TTA GAT CTC CAG TCG TGT CCC CCC TCC GAA CGT-3′

Gel-purified PCR products for hPAM4 V_(K) were restriction-digested withPvuII and BglII and cloned into the complementary PvuII/Bc10 sites ofthe light chain staging vector, V_(K)pBR2. V_(K)pBR2 is a modifiedstaging vector of V_(K)pBR (Leung et al., Hybridoma, 13:469, 1994), intowhich a XbaI restriction site was introduced at sixteen bases upstreamof the translation initiation codon. The assembled Vic genes weresubcloned as XbaI-BamHI restriction fragments into the expression vectorcontaining the V_(H) sequence, hPAM4 V_(H)pdHL2. The resultingexpression vectors were designated as hPAM4pdHL2.

Approximately 30 μg of hPAM4pdHL2 was linearized by digestion with SalIand transfected into Sp2/0-Ag14 cells by electroporation at 450 V and 25μF. The transfected cells were plated into 96-well plates and incubatedin a CO₂ cell culture incubator for two days and then selected for MTXresistance. Colonies surviving selection emerged in two to three weeksand were screened for human antibody secretion by ELISA assay. Briefly,supernatants (˜100 ul) from the surviving colonies were added into thewells of an ELISA microplate precoated with goat anti-human IgG F(ab′)₂fragment-specific Ab. The plate was incubated for one hour at roomtemperature. Unbound proteins were removed by washing three times withwash buffer (PBS containing 0.05% Tween-20). Horseradishperoxidase-conjugated goat anti-human IgG Fc fragment-specific Ab wasadded to the wells. Following incubation for one hour, a substratesolution (100 μL/well) containing 4 mM o-phenylenediaminedihydrochloride (OPD) and 0.04% H₂O₂ in PBS was added to the wells afterwashing. Color was allowed to develop in the dark for 30 minutes and thereaction was stopped by the addition of 50 μL of 4 NH₂SO₄ solution. Thebound human IgG was measured by reading the absorbance at 490 nm on anELISA reader. Positive cell clones were expanded and hPAM4 was purifiedfrom cell culture supernatant by affinity chromatography on a Protein Acolumn.

The Ag-binding activity of hPAM4 was confirmed by ELISA assay in amicrotiter plate coated with pancreas cancer cell extracts. An ELISAcompetitive binding assay using PAM4-antigen coated plates was developedto assess the Ag-binding affinity of hPAM4 in comparison with that of achimeric PAM4 composed of murine V and human C domains. Constant amountsof the HRP-conjugated cPAM4 mixed with varying concentrations of cPAM4or hPAM4 were added to the coated wells and incubated at roomtemperature for 1-2 h. The amount of HRP-conjugated cPAM4 bound to theCaPan1 Ag was revealed by reading the absorbance at 490 nm after theaddition of a substrate solution containing 4 mM o-phenylenediaminedihydrochloride and 0.04% H₂O₂. As shown by the competition assays inFIG. 4, hPAM4 and cPAM4 antibodies exhibited similar binding activities.

Example 2 Immunohistochemistry Staining Studies

Immunohistochemistry on normal adult tissues showed that the PAM4reactive epitope was restricted to the gastrointestinal tract wherestaining was weak, yet positive (Table 2). Normal pancreatic tissue,including ducts, ductules, acini, and islet cells, were negative forstaining. A PAM4 based enzyme immunoassay with tissue homogenates asantigens generally supported the immunohistology data (Table 3). ThePAM4 epitope was absent from normal pancreas and othernon-gastrointestinal tissues. In neoplastic tissues, PAM4 was reactivewith twenty one out of twenty five (85%) pancreatic cancers (Table 4 andTable 5) and ten out of twenty six colon cancers, but only limitedreactivity with tumors of the stomach, lung, breast, ovary, prostate,liver or kidney (Table 5). PAM4 reactivity appeared to correlate withthe stage of tumor differentiation, with a greater percentage ofstaining observed in well differentiated pancreatic cancers than inmoderately differentiated or poorly differentiated tumors. Generally,poorly differentiated tumors represent less than 10% of all pancreaticcancers.

These studies have shown the PAM4 reactivity and tissue distribution(both normal and cancer) to be unlike that reported for the CA19.9,DUPAN2, SPAN1, Nd2 and B72.3 antibodies and antibodies against the Lewisantigens. Together with crossblocking studies performed with certain ofthese MAbs, the data suggests that the PAM4 MAb recognizes a unique andnovel epitope. When compared to the antigens recognized by the CA19.9,DUPAN2, and anti-Le^(a) antibodies, the PAM4 antigen appears to be morerestricted in its tissue distribution and is reactive with a higherpercentage of pancreatic tumors. Moreover, it gives a greater overallintensity of reaction at equivalent concentrations and is reactive witha higher percentage of cells within the pancreatic tumors. Finally, PAM4was found to be only weakly reactive with three out of twelve chronicpancreatitis specimens, whereas CA19.9 and DUPAN2 were strongly reactivewith all twelve specimens. Although it is recognized that specificity isdependent in part upon the type of assay employed and the range andnumber of tissues examined, the ability of PAM4 to discriminate betweennormal and neoplastic pancreatic tissue, its ability to react with alarge percentage of the cancer specimens, the high intensity of thereactions, and the ability to distinguish between early stage pancreaticcancer and benign conditions such as pancreatitis are importantcharacteristics of this exemplary anti-pancreatic cancer antibody.

TABLE 2 Immunoperoxidase Staining of Normal Adult Tissues with MAb PAM4Staining Tissue Reaction Pancreas (22)^(a) − Ducts − Acini − Islets −Submaxillary gland (2) − Esophagus (2) − Stomach (3) +mucus secretingcells Duodenum (3) +goblet cells Jejunum (3) +goblet cells Ileum (3)+goblet cells Colon (5) +goblet cells Liver (3) − Gallbladder (2) −Bronchus (3) − Lung (3) − Heart (3) − Spleen (3) − Kidney (3) − Bladder(3) − Prostate (2) − Testes (2) − Uterus (2) − Ovary (2) − ^(a)number ofindividual specimens examined in parentheses

TABLE 3 Monoclonal Antibody PAM4 Reactivity with Normal Adult TissueHomogenates by EIA Tissue μg/g tissue^(a) Pancreas 6.4 Esophagus 8.1Stomach 61.3 Duodenum 44.7 Jejunum 60.6 Colon 74.5 Liver 0.0 Gallbladder5.6 Heart 3.7 Spleen 3.4 Kidney 6.6 Bladder 4.9 Thyroid 3.5 Adrenal 1.3Ureter 2.6 Testes 3.9 CaPan1 Pancreatic Tumor 569 ^(a)values are meanfrom two autopsy specimens

TABLE 4 Immunohistochemical Reactivity of Several Monoclonal Antibodieswith Pancreatic Tumors Differentiation PAM4 CA19.9 Le^(a) DUPAN2 1 W +++− − +++ 2 M ++ +++ +++ + 3 M + − + + 4 M +++ +++ +++ + 5 M ++ + − − 6M + ND ND ND 7 M* +++ +++ +++ +++ 8 M + − − +++ 9 M ++ + ++ − 10 M* ++++ ++ +++ 11 M ++ +++ +++ + 12 M ++ + + +++ 13 M + +++ +++ + 14 M ++ + +++ 15 M +++ + + ++ 16 M + + ++ − 17 M − + + − 18 M ++ ++ ++ ++ 19 M+++ + +++ ++ 20 M + − − − 21 M +++ +++ + ++ 22 P + + + +++ 23 P − − − −24 P − − − − 25 P − − + − TOTAL 21/25 17/24 18/24 16/24 −: Negative; +:5-20% of tissue is stained; ++: 21-50% of tissue is stained; +++: >50%of tissue is stained; W, M, P: Well, moderate, or poor differentiation;*Metastatic tissue; ND: Not Done

TABLE 5 Immunoperoxidase Staining of Neoplastic Tissues with MAb PAM4Cancer Tissue Positive/Total Pancreas 21/25 Colon 10/26 Stomach 1/5 Lung 1/15 Breast  0/30 Ovarian  0/10 Prostate 0/4 Liver  0/10 Kidney 0/4

Example 3 In Vivo Biodistribution and Tumor Targeting of RadiolabeledPAM4

Initial biodistribution studies of PAM4 were carried out in a series offour different xenografted human pancreatic tumors covering the range ofexpected differentiation. Each of the four tumor lines employed, AsPc1,BxPc3, Hs766T and CaPan1, exhibited concentrations of ¹³¹I-PAM4 withinthe tumors (range: 21%-48% ID/g on day three) that were significantly(P<0.01-0.001) higher than concomitantly administered nonspecific,isotype-matched Ag8 antibody (range: 3.6%-9.3% ID/g on day three). Thebiodistribution data were used to estimate potential radiation doses tothe tumor of 12,230; 10,684; 6,835; and 15,843 cGy/mCi of injected doseto AsPc1, BxPc3, Hs766T and CaPanl, respectively. With an actual maximumtolerated dose (MTD) of 0.7 mCi, PAM4 could provide substantial rad doseto each of the xenografted tumor models. In each tumor line the bloodlevels of radiolabeled PAM4 were significantly (P<0.01-0.001) lower thanthe nonspecific Ag8. Potential radiation doses to the blood from PAM4were 1.4-4.4 fold lower than from Ag8. When radiation doses to the tumorfrom PAM4 were normalized to the blood doses from PAM4, the tumorsreceived doses that were 2.2; 3.3; 3.4; and 13.1-fold higher than blood,respectively. Importantly, potential radiation doses to non-tumortissues were minimal.

The biodistribution of PAM4 was compared with an anti-CEA antibody,MN-14, using the CaPan1 tumor model. The concentration of PAM4 withinthe tumor was much greater than MN-14 at early timepoints, yieldingtumor:blood ratios at day three of 12.7±2.3 for PAM4 compared to 2.7±1.9for MN-14. Although PAM4 uptake within the tumor was significantlyhigher than for MN-14 at early timepoints (day one—P<0.001; daythree—P<0.01), dosimetry analyses indicated only a 3.2-fold higher doseto the tumor from PAM4 as compared to MN-14 over the fourteen day studyperiod. This was due to a rapid clearance of PAM4 from the tumor, suchthat at later timepoints similar concentrations of the two antibodieswere present within the tumors. A rapid clearance of PAM4 from the tumorwas also noted in the BxPc3 and Hs766T but not AsPc1 tumor models. Theseobservations were unlike those reported for other anti-mucin antibodies,as for example G9 and B72.3 in colorectal cancer, where each exhibitedlonger retention times as compared to the MN-14 antibody. Results fromstudies on the metabolism of PAM4, indicate that after initial bindingto the tumor cell, antibody is rapidly released, possibly beingcatabolized or being shed as an antigen:antibody complex. The bloodclearance is also very rapid. These data suggest that ¹³¹I may not bethe appropriate choice of isotope for therapeutic applications. Ashort-lived isotope, such as ⁹⁰Y or ¹⁸⁸Re, which can be administeredfrequently may be a more effective reagent.

PAM4 showed no evidence of targeting to normal tissues, except in theCaPan1 tumor model, where a small but statistically significant splenicuptake was observed (range 3.1-7.5% ID/g on day-3). This type of splenictargeting has been observed in the clinical application of theanti-mucin antibodies B72.3 and CC49. Importantly, these studies alsoreported that splenic targeting did not affect tumor uptake of antibodynor did it interfere with interpretation of the nuclear scans. Thesestudies suggested that splenic targeting was not due to crossreactiveantigens in the spleen, nor to binding by Fc receptors, but rather toone or more of the following possibilities: direct targeting of antigentrapped in the spleen, or indirect uptake of antigen:antibody complexesformed either in the blood or released from the tumor site. The latterwould require the presence of immune complexes in the blood. However,these were not observed when specimens as early as five minutes and aslate as seven days were examined by gel filtration (HPLC, GF-250column); radiolabeled antibody eluted as native material. The formerexplanation seems more likely in view of the fact that the CaPan1 tumorproduced large quantities of PAM4-reactive antigen, 100- to 1000-foldhigher than for the other tumor cell lines examined. The lack of splenictargeting by PAM4 in these other tumor lines suggests that thisphenomenon was related to excessive antigen production. Splenictargeting can be overcome by increasing the protein dose to 10 μg fromthe original 2 μg dose. A greater amount of the splenic entrappedantigen presumably was complexed with unlabeled PAM4 rather thanradiolabeled antibody. Increasing the protein dose had no adverse effectupon targeting of PAM4 to the tumor or nontumor tissues. In fact, anincrease of the protein dose to 100 μg more than doubled theconcentration of radiolabeled PAM4 within the CaPan1 tumor.

Example 4 Development of Orthotopic Pancreatic Tumor Model in AthymicNude Mice

In order to resemble the clinical presentation of pancreatic cancer inan animal model more closely, we developed an orthotopic model byinjecting tumor cells directly into the head of the pancreas. OrthotopicCaPan1 tumors grew progressively without overt symptoms until thedevelopment of ascites and death at ten to fourteen weeks. By three tofour weeks post-implantation, animals developed a palpable tumor ofapproximately 0.2 g. Within eight weeks of growth, primary tumors ofapproximately 1.2 g along with metastases to the liver and spleen wereobserved (1-3 metastatic tumors/animal; each tumor <0.1 g). At ten tofourteen weeks seeding of the diaphragm with development of ascites wereevident. Ascites formation and occasional jaundice were usually thefirst overt indications of tumor growth. At this time tumors were quitelarge, 1 to 2 g, and animals had at most only three to four weeks untildeath occurred.

Radiolabeled ¹³¹I-PAM4, administered to animals bearing four week oldorthotopic tumors (approximately 0.2 g) showed specific targeting to theprimary tumor with localization indices of 7.9±3.0 at day one increasingto 22.8±15.3 at day fourteen. No evidence of specific targeting to othertissues was noted. In one case where tumor metastases to the liver andspleen were observed, both metastases were targeted, and had highconcentrations of radiolabeled antibody. In addition, approximately halfof the animals developed a subcutaneous tumor at the incision site. Nosignificant differences were noted in the targeting of orthotopic andsubcutaneous tumors within the same animal, and no significantdifferences were observed in the targeting of orthotopic tumor whetheror not the animal had an additional subcutaneous tumor. The estimatedradiation doses from PAM4 were 6,704 and 1,655 cGy/mCi to the primarytumor and blood, respectively.

Example 5 Experimental Radioimmunotherapy of Pancreatic Cancer

The initial studies on the use of ¹³¹I-PAM4 for therapy were carried outwith the CaPan1 tumor, which was grown as a subcutaneous xenograft inathymic mice. Animals bearing a 0.25 g tumor were administered 350 μCi,¹³¹I-PAM4 in an experiment that also compared the therapeutic effects ofa similar dose of nonspecific Ag8. The MTD for administration of¹³¹I-PAM4 to animals bearing 1 cm³ tumors is 700 μCi. By weeks five andsix, the PAM4 treated animals showed a dramatic regression of tumor, andeven at week twenty seven, five out of eight remained tumor free. Theuntreated, as well as Ag8-treated animals, showed rapid progression oftumor growth although a significant difference was noted between thesetwo control groups. At seven weeks, tumors from the untreated group hadgrown 20.0±14.6-fold from the initial timepoint whereas the¹³¹I—Ag8-treated tumors had grown only 4.9±1.8-fold. At this time point,the PAM4 tumors had regressed to 0.1±0.1-fold of their original size, asignificant difference from both untreated (P<0.001) and nonspecificAg8-treated (P <0.01) animals.

These data show that CaPan1 tumors were sensitive to treatment with¹³¹I-PAM4. The outcome, that is, regression or progression of the tumor,was dependent upon several factors including initial tumor size. Thus,groups of animals bearing CaPan1 tumor burdens of 0.25 g, 0.5 g, 1.0 g,or 2.0 g were treated with a single dose of the 350 μCi ¹³¹I-PAM4. Themajority of animals having tumors of initial size 0.25 g and 0.5 g (nineof ten animals in each group) showed tumor regression or growthinhibition for at least sixteen weeks post treatment. In the 1.0 g tumorgroup five out of seven showed no tumor growth for the sixteen weekperiod and in the 2.0 g tumor group six out of nine showed no tumorgrowth for a period of six weeks before progression occurred. Although asingle 350 pEi dose was not as effective against larger tumors, a singledose may not be the appropriate regimen for large tumors.

Toxicity studies indicate the ability to give multiple cycles ofradioimmunotherapy, which may be more effective with a larger tumorburden. Animals bearing CaPan1 tumors averaging 1.0 g, were given eithera single dose of 350 μCi ¹³¹I-PAM4, two doses given at times zero andfour weeks or were left untreated. The untreated group had a meansurvival time of 3.7±1.0 weeks (survival defined as time for tumor toreach 5 cm³). Animals died as early as three weeks, with no animalsurviving past six weeks. A single dose of 350 μCi ¹³¹I-PAM4 produced asignificant increase in the survival time to 18.8±4.2 weeks (P<0.0001).The range of animal deaths extended from weeks thirteen to twenty five.None of the animals were alive at the end of the study period of twentysix weeks.

A significant increase in survival time was observed for the two dosegroup as compared to the single dose group. Half of the animals werealive at the twenty six week timepoint with tumor sizes from 1.0-2.8cm³, and a mean tumor growth rate of 1.6±0.7 fold from initial tumorsize. For those animals that were non-survivors at twenty six weeks, themean survival time (17.7±5.3 weeks) was similar to the single dosegroup.

Therapy studies with PAM4 were also conducted using the orthotopic tumormodel. Groups of animals bearing four week old orthotopic tumors(estimated tumor weight of 0.25 g) were either left untreated or treatedwith a single dose of either 350 μCi ¹³¹I-PAM4 or 350 of¹³¹I-nonspecific Ag8. The untreated animals had a 50% death rate by weekten with no survivors at week fifteen. Animals administered nonspecific¹³¹I—Ag8 at four weeks of tumor growth, showed a 50% death rate at weekseven with no survivors at week fourteen. Although statistically(logrank analysis) there were no differences between these two groups,it is possible that radiation toxicity had occurred in the Ag8 treatedanimals. Radiolabeled PAM4 provided a significant survival advantage(P<0.001) as compared to the untreated or Ag8 treated animals, with 70%survival at sixteen weeks, the end of the experiment. At this time thesurviving animals were sacrificed to determine tumor size. All animalshad tumor with an average weight of 1.2 g, as well as one or two small(<0.1 g) metastases evident in four of the seven animals. At sixteenweeks of growth, these tumors were more representative of aneight-week-old tumor.

Example 6 Combined Modality GEMZAR® Chemotherapy and ¹³¹I-PAM4Experimental Radioimmunotherapy

Initial studies into the combined use of gemcitabine (GEMZAR®) with¹³¹I-PAM4 radioimmunotherapy were performed as a checkerboard array; asingle dose of gemcitabine (0, 100, 200, 500 mg/kg) versus a single doseof ¹³¹I-PAM4 ([MTD=700 μCi] 100%, 75%, 50%, 0% of the MTD). The combinedMTD was found to be 500 mg/kg gemcitabine with 350 μCi ¹³¹I-PAM4 (50%MTD). Toxicity, as measured by loss of body weight, went to the maximumconsidered as nontoxic; that is 20% loss in body weight. Although thecombined treatment protocol was significantly more effective thangemcitabine alone, the treatment was no more effective thanradioimmunotherapy alone. The next studies were performed at a low doseof gemcitabine and radioimmunotherapy to examine if a true synergistictherapeutic effect would be observed. Athymic nude mice bearing tumorsof approximately 1 cm³ (approximately 5% of body weight) wereadministered gemcitabine, 100 mg/kg on days zero, three, six, nine, andtwelve, with 100 μCi of ¹³¹I-PAM4 given on day zero. A therapeuticeffect was observed with statistically significant (P<0.0001) regression(two of five tumors less than 0.1 cm³) and/or growth inhibition of thetumors compared to gemcitabine alone. Thus, at lower dosages oftherapeutic agent, there surprisingly appears to be a synergistic effectof the combination of gemcitabine and radioimmunotherapy. Of additionalnote, in terms of body weight, toxicity was not observed. Thecombination treatment protocol can, if necessary, be delivered inmultiple cycles, with the second treatment cycle beginning in week-four,as was done with the radioimmunotherapy-alone studies described above.

Example 7 Binding of PAM4 Antibodies to Transfected Cell Lines

Transfected Pancreatic Cells

PanC1 human pancreatic adenocarcinoma cells that do not express theMUC-1 mucin were transfected with MUC-1 encoding cDNA as disclosed inHudson et al. (Amer. J. Pathol. 148, 3:951-60, 1996) and obtained fromDr. M. A. Hollingsworth (Univ. of Nebraska Medical Center, Omaha,Nebr.). The MUC-1 cDNAs encoded either 30 tandem repeat (30TR) or 42tandem repeat (42TR) versions of MUC-1 (Hudson et al., 1996; Lidell etal, FEBS J. 275:481-89, 2008). The MUC-1 sequences were identical otherthan in the number of tandem repeat sequences encoded.

PanC1 cells transfected with either 30TR or 42TR MUC-1 or controlvectors (no insert or reversed insert) or untransfected PanC1 cells wereexamined for reactivity with PAM4 antibody by enzyme immunoassay of thesupernatants from cell culture. Neither the untransfected PanC1 cellsnor the control transfected PanC1 cells produced detectable levels ofPAM4-reactive mucins by immunoassay (not shown). However, both the 30TRand 42TR MUC-1 transfected cells were highly reactive with PAM4 antibody(not shown).

The transfected PanC1 cells were reexamined using a more sensitiveimmunoassay. Briefly, cells were grown in T75 flasks until they reachedapproximately 80%-90% confluency (˜4-5 days from initial seeding). Atthis time, the spent-media were collected, centrifuged at high speed,and used for quantitation of PAM4-reactive mucin by enzyme immunoassay.The cells were also collected and counted. Both the Panc1 parent cellline originating from Dr. Hollingsworth and a separate Panel cell lineobtained from the American Type Culture Collection (Manassas, Va.), aswell as the vector control, produced low, but detectable quantities ofPAM4-reactive mucin (0.87±0.17, 0.54±0.17 and 0.02±0.02 μg/mL/10⁶ cells,respectively), whereas the 30TR-MUC-1 gene transfected cells produced14.17*2.22 μg/mL/10⁶ cells (P<0.0003 or better for comparison of30TR-MUC-1 gene transfected media compared to all other samples).

It has been reported that transfection of PanC1 cells with MUC-1 cDNA,in addition to increasing expression of MUC-1 by the transfected PanC1cells, also has secondary effects on cell protein expression, such asincreasing levels of cytokeratins 8 and 18 in the transfected cells(Hudson et al., 1996, Abstract), but only in cells transfected with thelarger (42TR) MUC-1 cDNA (Hudson et al. 1996, pg. 956, col. 1, 3^(rd)paragraph).

Transfected Kidney Cells

MUC-1 gene-transfected HEK-293 (human embryonic kidney cells) producedMUC-1 that was reactive with the MA5 monoclonal antibody, but that wasnot reactive with PAM4 (not shown). However, as discussed above,MUC-1-transfected PanC1 cells that express very low levels of endogenousMUC-1 synthesized MUC-1 that was strongly reactive with PAM4 andnon-reactive with MAb-MA5. Heterologous sandwich immunoassays (PAM4→MA5and MA5→PAM4 capture/probe) did not function to produce a signal withsupernatants from several cell lines. Use of a polyclonal anti-mucinantiserum as probe with the PAM4 or MA5 MAbs as capture reagents didprovide effective immunoassays. Cross-blocking of these two MAbs withintheir respective immunoassays using the polyclonal as the probesuggested these MAbs were reactive with independent epitopes.

The data suggest that the PAM4 and MA5 epitopes are not co-expressedwithin the same antigenic molecule, and that while the PanC1 cell linemay possess biosynthetic processes that create the PAM4-epitope, theHEK-293 cells do not. Differences in post-translational modification ofthe MUC-1 protein core (expression/activity of specificglycosyltransferases) may be responsible for these findings.

Example 8 Effects of Reagent Treatment on Immunoreactivity of PAM4Antigen

Treatment of pancreatic mucin PAM4 antigen with DTT (15 min at roomtemp), completely abolished reactivity with PAM4 (DTT-EC₅₀, 0.60±0.00μM). The only cysteines (cystine bridges) within MUC-1 are presentwithin the transmembrane domain and should not be accessible to DTT. Thesecreted form of MUC-1 does not contain the transmembrane domain andtherefore has no intramolecular cystine bridges. Data from periodateoxidation treatment of PAM4 antigen with 0.05 M sodium periodate for 2hrs at room temperature yielded 40% loss of immunoreactivity with PAM4antibody (not shown). Further periodate studies have shown as high as a60% loss of immunoreactivity with PAM4 antibody (not shown). The resultsof periodate and DTT studies suggest that the PAM4 epitope isconformationally dependent upon some minimal form of glycosylation, andmay be affected by intermolecular disulfide bond formation.

Example 9 Distribution and Cross-Reactivity of the PAM4 Antigen

The expression of the PAM4-epitope within PanINs is atypical for MUC-1.It is similar to the expression reported for MUC-5ac as detected by thecommercially available MAb-CLH2-2. However, an attempted sandwichimmunoassay with PAM4 capture and MAb-CLH2-2 as probe gave negativeresults. Although this possibly suggests the PAM4 and CLH2-2 epitopesmay overlap and thus block each other, the CLH2-2 was reported to bereactive with 42/66 (64%) gastric carcinomas whereas the PAM4 MAb showedreactivity with only 6/40 (15%) of gastric carcinomas and, of these,only in focal reactivity.

Use of the commercially available 45M1, an anti-MUC-5ac MAb, as a probereagent in EIA (with PAM4 as capture) provided positive results,indicating that the two epitopes may be present on the same antigenicmolecule. Blocking studies (either direction) indicated that theepitopes bound by 45M1 and PAM4 are in fact two distinct epitopes, as noblocking was observed. Labeling of tissue microarrays consisting ofcores from invasive pancreatic carcinoma has demonstrated significantdifferences for expression of the 45M1 and PAM4 epitopes in individualpatient specimens. Of 28 specimens, concordance was observed in only 17cases (61%). PAM4 was reactive with 24/28 cases (86%) while 45M1 wasreactive with 13/28 (46%) cases (not shown).

It is possible that in the studies above (Example 7) regarding MUC-1gene transfection, expression of MUC-1 may have upregulated expressionof another mucin, or affected exposure of the PAM4-epitope in some othermanner. The results of periodate studies are consistent withglycosylation as a factor in PAM4 antigen immunoreactivity with the PAM4antibody. Thus, results of studies with apomucins may not be definitivefor antigen determination.

Although based on EIA capture, the PAM4 antibody appears to bind to thesame antigenic protein as the 45M1 anti-MUC-5ac MAb, it is noted thatMUC-5ac is not specific to pancreas cancer and it is found in a numberof normal tissues (other than the gastric mucosa with which PAM4 isreactive). For example, MUC-5ac is found in normal lung, colon and othertissues. PAM4 antibody does not bind to normal lung tissues, except asindicated above in few samples and to a limited or minimal amount.

With respect to the effects of DTT and periodate, it is probable thatthe peptide core disulfide bridges are identical no matter what tissueproduces the protein. A specific amino acid sequence should fold in aspecific manner, independent of the tissue source. However,glycosylation patterns may differ dependent upon tissue source.

Example 10 Phage Display Peptide Binding of PAM4 Antibody

PAM4 antibody binding was examined with two different phage displaypeptide libraries. The first was a linear peptide library consisting of12 amino acid sequences and the second was a cyclic peptide consistingof 7 amino acid sequences cyclized by a disulfide bridge. We panned theindividual libraries alternately against hPAM4 and hLL2 (negativeselection with anti-CD22 antibody) for a combined total of 4 rounds, andthen screened the phage displayed peptide for reactivity with both hPAM4and mPAM4 with little to no reactivity against hLL2. Phage binding in anon-specific manner (i.e., binding to epratuzumab [hLL2]) werediscarded.

For the linear phage-displayed peptide, the sequence WTWNITKAYPLP (SEQID NO:7) was identified 30 times (in 35 sequenced phage), each of whichwere shown to have reactivity with PAM4 antibodies. A mutationalanalysis was conducted in which a library based on this sequence andhaving 7.5% degeneracy at each position, was constructed, panned andscreened as before. Variability was noted in the 19 obtained peptidesequences that were positive for PAM4 binding with 7 being identical tothe parental sequence, 5 having the sequence WTWNITKEYPQP (SEQ ID NO:62)and the rest being uniquely present. Table 6 shows the results of thismutational analysis. The upper row lists the sequences identified andthe lower row lists the frequency with which each of the amino acids wasidentified in that position. The parent sequence is most frequent (bold)with the next highest variation a substitution of E for A at position 8and a substitution of Q for L at position 11. It does not appear thatthese substitutions had any great effect upon immunoreactivity.

TABLE 6 Phage Display Amino Acid Sequence Variation with Linear PeptideBinding to PAM4 Antibody W T W N I T K A Y P L P D R R E T R Q T T I N RM G F C C number of 19 19 19 18 19 17 14 10 18 17 11 19 occurrences 1 21 5 1 2 5 (out of 19 2 1 1 sequences 1 1 1 analyzed) 1 1 1 1

Results with the phage displayed cyclic library were significantlydifferent from the linear library (Table 7). The sequence ACPEVVWGTTC(SEQ ID NO:63) was present in 33 of 35 peptide sequences examined.Analysis of the cyclic library presented the following results(positions with an asterisk were invariant and not subject to selectivepressure in the library).

TABLE 7 Phage Display Amino Acid Sequence Variation with Linear PeptideBinding to PAM4 Antibody A C P E W W G T T C Y S G M S S Q P numberof * * 33 35 35 35 34 29 28 * occurrences 2 1 5 4 (out of 19 1 1sequences 1 analyzed) 1

The two cysteines (at positions 2 and 10) formed a disulfide bridge.Substitution of T at position 9 with any amino acid greatly affectedimmunoreactivity. The sequence GTTGTTC (SEQ ID NO:64) is present withinthe MUC-5ac protein towards the amino terminus as compared to the cyclicpeptide sequence shown above, which shows homology at the C-terminal endof the consensus peptide sequence. However, the cyclic peptide onlyshowed approximately 10% of the immunoreactivity of the linear sequencewith the PAM4 antibody. Both linear and cyclic consensus sequences areassociated with a cysteine, which may or may not relate to the effect ofDTT on PAM4 antigen immunoreactivity.

The results reported herein indicate that the PAM4 epitope is dependentupon a specific conformation which may be produced by disulfide bridges,as well as a specific glycosylation pattern.

Example 11 Immunohistology of Pancreatic Cancer in a PancreatitisSpecimen

Several pathologic conditions predispose patients to the development ofpancreatic carcinoma, such as pancreatitis, diabetes, smoking andothers. Within this pre-selected group of patients, screening measuresare particularly important for the early detection of pancreaticneoplasia. We examined 9 specimens of chronic pancreatitis tissue frompatients having primary diagnosis of this disease. We employed ananti-CD74 MAb, LL1, as an indicator of inflammatory infiltrate, andMAb-MA5 as a positive control for pancreatic ductal and acinar cells.Whereas the two control MAbs provided immunohistologic evidenceconsistent with pancreatitis, in no instance did PAM4 react withinflamed pancreatic tissue. However, in one case, a moderatelydifferentiated pancreatic adenocarcinoma was also present within thetissue specimen. PAM4 gave an intense stain of the neoplastic cellswithin this tumor. In a second case, while the inflamed tissue wasnegative with PAM4, a small PanIN precursor lesion was identified thatwas labeled with PAM4. Labeling of the PanIN within this latter specimenis consistent with early detection of pancreatic neoplasia in a patientdiagnosed with a non-malignant disease. These results show thatdetection and/or diagnosis using the PAM4 antibody may be performed withhigh sensitivity and selectivity for pancreatic neoplasia against abackground of benign pancreatic tissues.

Example 12 Therapy of a Patient With Inoperable and MetastaticPancreatic Carcinoma

Patient 118-001, CWG, is a 63-year-old man with Stage-IV pancreaticadenocarcinoma with multiple liver metastases, diagnosed in November of2007. He agreed to undertake combined radioimmunotherapy and gemcitabinechemotherapy as a first treatment strategy, and was then given a firsttherapy cycle of 6.5 mCi/m² of ⁹⁰Y-hPAM4, combined with 200 mg/m²gemcitabine, whereby the gemcitabine was given once weekly on weeks 1-4and ⁹⁰Y-hPAM4 was given once-weekly on weeks 2-4 (3 doses). Two monthslater, the same therapy cycle was repeated, because no major toxicitieswere noted after the first cycle. Already 4 weeks after the firsttherapy cycle, CT evidence of a reduction in the diameters of theprimary tumor and 2 of the 3 liver metastases surprisingly was noted,and this was consistent with significant decreases in the SUV values ofFDG-PET scans, with 3 of the 4 tumors returning to normal background SUVlevels at this time (FIG. 6 and FIG. 7). The patient's pre-therapyCA-19.9 level of 1,297 dropped to a low level of 77, further supportiveof the therapy being effective. Table 8 shows the effects of combinedradioimmunotherapy with ⁹⁰Y-hPAM4 and gemcitabine chemotherapy in thispatient. It was surprising and unexpected that such low doses of theradionuclide conjugated to the antibody combined with such low,nontoxic, doses of gemcitabine showed such antitumor activity even afteronly a single course of this therapy.

TABLE 8 Effects of Combined Radioimmunotherapy with ⁹⁰Y-hPAM4 andGemcitabine Chemotherapy in Metastatic Pancreatic Carcinoma Baseline 4wk post-Tx Longest Longest Baseline Tumor Diameter Diameter PET 4 wkpost-Tx Location (cm) (cm) (SUV) PET (SUV) Pancreatic tail 4.5 4.3 9.24.2 (primary) L hepatic met 1.9 1.9 4.1 background R post hepatic 1.71.6 3.7 background met R central hepatic 1.9 1.2 3.2 background met

Example 13 Therapy of a Patient with Inoperable Metastatic PancreaticCarcinoma

A 56-year-old male with extensive, inoperable adenocarcinoma of thepancreas, with several liver metastases ranging from 1 to 4 cm indiameter, substantial weight loss (30 lbs of weight or more), mildjaundice, lethargy and weakness, as well as abdominal pains requiringmedication, is given 4 weekly infusions of gemcitabine at doses of 200mg/m² each. On the last three gemcitabine infusions, ⁹⁰Y-DOTA-hPAM4radiolabeled humanized antibody is administered at a dose of 10 mCi/m²of ⁹⁰Y and 20 mg antibody protein, in a two-hour i.v. infusion. Twoweeks later, the patient is given a course of gemcitabine chemotherapyconsisting of 3 weekly doses of 600 mg/m² by i.v. infusion. The patientis then evaluated 4 weeks later, and has a mild leukopenia (grade-2), noother major blood or enzyme changes over baseline, but shows animprovement in the blood CA19.9 titer from 5,700 to 1,200 and a decreasein jaundice, with an overall subjective improvement. This follows 3weeks later with a repeat of the cycle of lower-dose gemcitabine (weeklyx 4), with 3 doses of ⁹⁰Y-DOTA-hPAM4. Four weeks later, the patient isreevaluated, and the CT and PET scans confirm an approximately 40%reduction of total tumor mass (primary cancer and metastases), with afurther reduction of the CA19.9 titer to 870. The patient regainsappetite and activity, and is able to return to more usual dailyactivities without the need for pain medication. He gains 12 lbs afterbeginning this experimental therapy. A repeat of the scans and bloodvalues indicates that this response is maintained 6 weeks later.

Example 14 Preparation of Dock-and-Lock (DNL) Constructs forPretargeting

DDD and AD Fusion Proteins

The DNL technique can be used to make dimers, trimers, tetramers,hexamers, etc. comprising virtually any antibodies or fragments thereofor other effector moieties. For certain preferred embodiments, IgGantibodies or Fab antibody fragments may be produced as fusion proteinscontaining either a dimerization and docking domain (DDD) or anchoringdomain (AD) sequence. Although in preferred embodiments the DDD and ADmoieties are produced as fusion proteins, the skilled artisan willrealize that other methods of conjugation, such as chemicalcross-linking or click chemistry, may be utilized within the scope ofthe claimed methods and compositions.

Bispecific antibodies may be formed by combining a Fab-DDD fusionprotein of a first antibody with a Fab-AD fusion protein of a secondantibody. Alternatively, constructs may be made that combine IgG-ADfusion proteins with Fab-DDD fusion proteins. The technique is notlimiting and any protein or peptide of use may be produced as an AD orDDD fusion protein for incorporation into a DNL construct. Wherechemical cross-linking is utilized, the AD and DDD conjugates are notlimited to proteins or peptides and may comprise any molecule that maybe cross-linked to an AD or DDD sequence using any cross-linkingtechnique known in the art. In certain exemplary embodiments, apolyethylene glycol (PEG) or other polymeric moiety may be incorporatedinto a DNL construct, as described in further detail below.

For pretargeting applications, an antibody or fragment containing abinding site for an antigen associated with a diseased tissue, such as atumor-associated antigen (TAA), may be combined with a second antibodyor fragment that binds a hapten on a targetable construct, to which atherapeutic and/or diagnostic agent is attached. The DNL-basedbispecific antibody is administered to a subject, circulating antibodyis allowed to clear from the blood and localize to target tissue, andthe conjugated targetable construct is added and binds to the localizedantibody for diagnosis or therapy.

Independent transgenic cell lines may be developed for each Fab or IgGfusion protein. Once produced, the modules can be purified if desired ormaintained in the cell culture supernatant fluid. Following production,any DDD-fusion protein module can be combined with any AD-fusion proteinmodule to generate a bispecific DNL construct. For different types ofconstructs, different AD or DDD sequences may be utilized. Exemplary DDDand AD sequences are provided below.

DDD1: (SEQ ID NO: 65) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2:(SEQ ID NO: 66) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1:(SEQ ID NO: 67) QIEYLAKQIVDNAIQQA AD2: (SEQ ID NO: 68)CGQIEYLAKQIVDNAIQQAGC

The skilled artisan will realize that DDD1 and DDD2 comprise the DDDsequence of the human RIIα form of protein kinase A. However, inalternative embodiments, the DDD and AD moieties may be based on the DDDsequence of the human RIα form of protein kinase A and a correspondingAKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.

DDD3 (SEQ ID NO: 69) SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEE AKDDD3C (SEQ ID NO: 70) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK AD3 (SEQ ID NO: 71) CGFEELAWKIAKMIWSDVFQQGC

Expression Vectors

The plasmid vector pdHL2 has been used to produce a number of antibodiesand antibody-based constructs. See Gillies et al., J Immunol Methods(1989), 125:191-202; Losman et al., Cancer (Phila) (1997), 80:2660-6.The di-cistronic mammalian expression vector directs the synthesis ofthe heavy and light chains of IgG. The vector sequences are mostlyidentical for many different IgG-pdHL2 constructs, with the onlydifferences existing in the variable domain (V_(H) and V_(L)) sequences.Using molecular biology tools known to those skilled in the art, theseIgG expression vectors can be converted into Fab-DDD or Fab-ADexpression vectors. To generate Fab-DDD expression vectors, the codingsequences for the hinge, CH₂ and CH3 domains of the heavy chain arereplaced with a sequence encoding the first 4 residues of the hinge, a14 residue Gly-Ser linker and the first 44 residues of human RIIα(referred to as DDD 1). To generate Fab-AD expression vectors, thesequences for the hinge, CH2 and CH3 domains of IgG are replaced with asequence encoding the first 4 residues of the hinge, a 15 residueGly-Ser linker and a 17 residue synthetic AD called AKAP-IS (referred toas AD1), which was generated using bioinformatics and peptide arraytechnology and shown to bind RIIα dimers with a very high affinity (0.4nM). See Alto, et al. Proc. Natl. Acad. Sci., U.S.A (2003), 100:4445-50.

Two shuttle vectors were designed to facilitate the conversion ofIgG-pdHL2 vectors to either Fab-DDD1 or Fab-AD1 expression vectors, asdescribed below.

Preparation of CH1

The CH1 domain was amplified by PCR using the pdHL2 plasmid vector as atemplate. The left PCR primer consisted of the upstream (5′) end of theCH1 domain and a SacII restriction endonuclease site, which is 5′ of theCH1 coding sequence. The right primer consisted of the sequence codingfor the first 4 residues of the hinge (PKSC) followed by four glycinesand a serine, with the final two codons (GS) comprising a Bam HIrestriction site. The 410 bp PCR amplimer was cloned into the PGEMT® PCRcloning vector (PROMEGA®, Inc.) and clones were screened for inserts inthe T7 (5′) orientation.

A duplex oligonucleotide, designated (G₄S)₂DDD1 ((G₄S)₂ disclosed as SEQID NO:37), was synthesized by Sigma GENOSYS® (Haverhill, UK) to code forthe amino acid sequence of DDD1 preceded by 11 residues of the linkerpeptide, with the first two codons comprising a BamHI restriction site.A stop codon and an EagI restriction site are appended to the 3′ end.The encoded polypeptide sequence is shown below.

(SEQ ID NO: 72) GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFT RLREARA

Two oligonucleotides, designated RIIA1-44 top and RIIA1-44 bottom, whichoverlap by 30 base pairs on their 3′ ends, were synthesized and combinedto comprise the central 154 base pairs of the 174 bp DDD1 sequence. Theoligonucleotides were annealed and subjected to a primer extensionreaction with Taq polymerase. Following primer extension, the duplex wasamplified by PCR. The amplimer was cloned into PGEMT® and screened forinserts in the T7 (5′) orientation.

A duplex oligonucleotide was synthesized to code for the amino acidsequence of AD1 preceded by 11 residues of the linker peptide with thefirst two codons comprising a BamHI restriction site. A stop codon andan EagI restriction site are appended to the 3′ end. The encodedpolypeptide sequence is shown below.

GSGGGGSGGGGSQIEYLAKQIVDNAIQQA (SEQ ID NO: 73)

Two complimentary overlapping oligonucleotides encoding the abovepeptide sequence, designated AKAP-IS Top and AKAP-IS Bottom, weresynthesized and annealed. The duplex was amplified by PCR. The amplimerwas cloned into the PGEMT® vector and screened for inserts in the T7(5′) orientation.

Ligating DDD1 with CH1

A 190 bp fragment encoding the DDD1 sequence was excised from PGEMT®with BamHI and NotI restriction enzymes and then ligated into the samesites in CH1-PGEMT® to generate the shuttle vector CH1-DDD1-PGEMT®.

Ligating AD1 with CH1

A 110 bp fragment containing the AD1 sequence was excised from PGEMT®with BamHI and NotI and then ligated into the same sites in CH1-PGEMT®to generate the shuttle vector CH1-AD1-PGEMT®.

Cloning CH1-DDD1 or CH1-AD1 into pdHL2-Based Vectors

With this modular design either CH1-DDD1 or CH1-AD1 can be incorporatedinto any IgG construct in the pdHL2 vector. The entire heavy chainconstant domain is replaced with one of the above constructs by removingthe SacII/EagI restriction fragment (CH1-CH3) from pdHL2 and replacingit with the SacII/EagI fragment of CH1-DDD1 or CH1-AD1, which is excisedfrom the respective pGemT shuttle vector.

Construction of h679-Fd-AD1-pdHL2

h679-Fd-AD1-pdHL2 is an expression vector for production of h679 Fabwith AD1 coupled to the carboxyl terminal end of the CH1 domain of theFd via a flexible Gly/Ser peptide spacer composed of 14 amino acidresidues. A pdHL2-based vector containing the variable domains of h679was converted to h679-Fd-AD1-pdHL2 by replacement of the SacII/EagIfragment with the CH1-AD1 fragment, which was excised from theCH1-AD1-SV3 shuttle vector with SacII and EagI.

Construction of C-DDD1-Fd-hMN-14-pdHL2

C-DDD1-Fd-hMN-14-pdHL2 is an expression vector for production of astable dimer that comprises two copies of a fusion proteinC-DDD1-Fab-hMN-14, in which DDD1 is linked to hMN-14 Fab at the carboxylterminus of CH1 via a flexible peptide spacer. The plasmid vectorhMN-14(I)-pdHL2, which has been used to produce hMN-14 IgG, wasconverted to C-DDD1-Fd-hMN-14-pdHL2 by digestion with SacII and EagIrestriction endonucleases to remove the CH1-CH3 domains and insertion ofthe CH1-DDD1 fragment, which was excised from the CH1-DDD1-SV3 shuttlevector with SacII and EagI.

The same technique has been utilized to produce plasmids for Fabexpression of a wide variety of known antibodies, such as hLL1, hLL2,hPAM4, hR1, hRS7, hMN-14, hMN-15, hA19, hA20 and many others. Generally,the antibody variable region coding sequences were present in a pdHL2expression vector and the expression vector was converted for productionof an AD- or DDD-fusion protein as described above. The AD- andDDD-fusion proteins comprising a Fab fragment of any of such antibodiesmay be combined, in an approximate ratio of two DDD-fusion proteins perone AD-fusion protein, to generate a trimeric DNL construct comprisingtwo Fab fragments of a first antibody and one Fab fragment of a secondantibody.

C-DDD2-Fd-hMN-14-pdHL2

C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for production ofC-DDD2-Fab-hMN-14, which possesses a dimerization and docking domainsequence of DDD2 appended to the carboxyl terminus of the Fd of hMN-14via a 14 amino acid residue Gly/Ser peptide linker. The fusion proteinsecreted is composed of two identical copies of hMN-14 Fab held togetherby non-covalent interaction of the DDD2 domains.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides, which comprise the coding sequence forpart of the linker peptide and residues 1-13 of DDD2, were madesynthetically. The oligonucleotides were annealed and phosphorylatedwith T4 PNK, resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases BamHI and PstI, respectively.

The duplex DNA was ligated with the shuttle vector CH1-DDD1-PGEMT®,which was prepared by digestion with BamHI and PstI, to generate theshuttle vector CH1-DDD2-PGEMT®. A 507 bp fragment was excised fromCH1-DDD2-PGEMT® with SacII and EagI and ligated with the IgG expressionvector hMN-14(I)-pdHL2, which was prepared by digestion with SacII andEagI. The final expression construct was designatedC-DDD2-Fd-hMN-14-pdHL2. Similar techniques have been utilized togenerated DDD2-fusion proteins of the Fab fragments of a number ofdifferent humanized antibodies.

h679-Fd-AD2-pdHL2

h679-Fab-AD2, was designed to pair as B to C-DDD2-Fab-hMN-14 as A.h679-Fd-AD2-pdHL2 is an expression vector for the production ofh679-Fab-AD2, which possesses an anchoring domain sequence of AD2appended to the carboxyl terminal end of the CH1 domain via a 14 aminoacid residue Gly/Ser peptide linker. AD2 has one cysteine residuepreceding and another one following the anchor domain sequence of AD1.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides (AD2 Top and AD2 Bottom), which comprisethe coding sequence for AD2 and part of the linker sequence, were madesynthetically. The oligonucleotides were annealed and phosphorylatedwith T4 PNK, resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases BamHI and SpeI, respectively.

The duplex DNA was ligated into the shuttle vector CH1-AD1-PGEMT®, whichwas prepared by digestion with BamHI and SpeI, to generate the shuttlevector CH1-AD2-PGEMT®. A 429 base pair fragment containing CH1 and AD2coding sequences was excised from the shuttle vector with SacII and EagIrestriction enzymes and ligated into h679-pdHL2 vector that prepared bydigestion with those same enzymes. The final expression vector ish679-Fd-AD2-pdHL2.

Example 15 Production of AD- and DDD-linked Fab and IgG Fusion ProteinsFrom Multiple Antibodies

Using the techniques described in the preceding Example, the IgG and Fabfusion proteins shown in Table 9 were constructed and incorporated intoDNL constructs. The fusion proteins retained the antigen-bindingcharacteristics of the parent antibodies and the DNL constructsexhibited the antigen-binding activities of the incorporated antibodiesor antibody fragments.

TABLE 9 Fusion proteins comprising IgG or Fab Fusion Protein BindingSpecificity C-AD1-Fab-h679 HSG C-AD2-Fab-h679 HSG C-(AD)₂-Fab-h679 HSGC-AD2-Fab-h734 Indium-DTPA C-AD2-Fab-hA20 CD20 C-AD2-Fab-hA20L CD20C-AD2-Fab-hL243 HLA-DR C-AD2-Fab-hLL2 CD22 N-AD2-Fab-hLL2 CD22C-AD2-IgG-hMN-14 CEACAM5 C-AD2-IgG-hR1 IGF-1R C-AD2-IgG-hRS7 EGP-1C-AD2-IgG-hPAM4 MUC C-AD2-IgG-hLL1 CD74 C-DDD1-Fab-hMN-14 CEACAM5C-DDD2-Fab-hMN-14 CEACAM5 C-DDD2-Fab-h679 HSG C-DDD2-Fab-hA19 CD19C-DDD2-Fab-hA20 CD20 C-DDD2-Fab-hAFP AFP C-DDD2-Fab-hL243 HLA-DRC-DDD2-Fab-hLL1 CD74 C-DDD2-Fab-hLL2 CD22 C-DDD2-Fab-hMN-3 CEACAM6C-DDD2-Fab-hMN-15 CEACAM6 C-DDD2-Fab-hPAM4 MUC C-DDD2-Fab-hR1 IGF-1RC-DDD2-Fab-hRS7 EGP-1 N-DDD2-Fab-hMN-14 CEACAM5

Example 16 Sequence variants for DNL

In certain preferred embodiments, the AD and DDD sequences incorporatedinto the DNL construct comprise the amino acid sequences of AD1, AD2,AD3, DDD1, DDD2, DDD3 or DDD3C as discussed above. However, inalternative embodiments sequence variants of AD and/or DDD moieties maybe utilized in construction of the DNL complexes. For example, there areonly four variants of human PKA DDD sequences, corresponding to the DDDmoieties of PKA RIα, RIIα, RIβ and RIIβ. The RIIα DDD sequence is thebasis of DDD1 and DDD2 disclosed above. The four human PKA DDD sequencesare shown below. The DDD sequence represents residues 1-44 of RIIα, 1-44of RIIβ, 12-61 of RIα and 13-66 of RIβ. (Note that the sequence of DDD1is modified slightly from the human PKA RIIα DDD moiety.)

PKA RIα (SEQ ID NO: 74) SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEAK PKA RIβ (SEQ ID NO: 75)SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEE NRQILA  PKA RIIα(SEQ ID NO: 76) SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ PKA RIIβ(SEQ ID NO: 77) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER

The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Hamuro et al., 2005,Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38;Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker etal., 2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al.,2006, Mol Ce1124:397-408, the entire text of each of which isincorporated herein by reference.)

For example, Kinderman et al. (2006) examined the crystal structure ofthe AD-DDD binding interaction and concluded that the human DDD sequencecontained a number of conserved amino acid residues that were importantin either dimer formation or AKAP binding, underlined in SEQ ID NO:65below. (See FIG. 1 of Kinderman et al., 2006, incorporated herein byreference.) The skilled artisan will realize that in designing sequencevariants of the DDD sequence, one would desirably avoid changing any ofthe underlined residues, while conservative amino acid substitutionsmight be made for residues that are less critical for dimerization andAKAP binding.

(SEQ ID NO: 65) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

Alto et al. (2003) performed a bioinformatic analysis of the AD sequenceof various AKAP proteins to design an RII selective AD sequence calledAKAP-IS (SEQ ID NO:67), with a binding constant for DDD of 0.4 nM. TheAKAP-IS sequence was designed as a peptide antagonist of AKAP binding toPKA. Residues in the AKAP-IS sequence where substitutions tended todecrease binding to DDD are underlined in SEQ ID NO:67. The skilledartisan will realize that in designing sequence variants of the ADsequence, one would desirably avoid changing any of the underlinedresidues, while conservative amino acid substitutions might be made forresidues that are less critical for DDD binding.

AKAP-IS sequence

QIEYLAKQIVDNAIQQA (SEQ ID NO: 67)

Gold (2006) utilized crystallography and peptide screening to develop aSuperAKAP-IS sequence (SEQ ID NO:78), exhibiting a five order ofmagnitude higher selectivity for the RII isoform of PKA compared withthe RI isoform. Underlined residues indicate the positions of amino acidsubstitutions, relative to the AKAP-IS sequence, which increased bindingto the DDD moiety of RIIα. In this sequence, the N-terminal Q residue isnumbered as residue number 4 and the C-terminal A residue is residuenumber 20. Residues where substitutions could be made to affect theaffinity for RIIα were residues 8, 11, 15, 16, 18, 19 and 20 (Gold etal., 2006). It is contemplated that in certain alternative embodiments,the SuperAKAP-IS sequence may be substituted for the AKAP-IS AD moietysequence to prepare DNL constructs. Other alternative sequences thatmight be substituted for the AKAP-IS AD sequence are shown in SEQ IDNO:79-81. Substitutions relative to the AKAP-IS sequence are underlined.It is anticipated that, as with the AD2 sequence shown in SEQ ID NO:68,the AD moiety may also include the additional N-terminal residuescysteine and glycine and C-terminal residues glycine and cysteine.

SuperAKAP-IS QIEYVAKQIVDYAIHQA (SEQ ID NO: 78)Alternative AKAP sequences QIEYKAKQIVDHAIHQA (SEQ ID NO: 79)QIEYHAKQIVDHAIHQA (SEQ ID NO: 80) QIEYVAKQIVDHAIHQA (SEQ ID NO: 81)

FIG. 2 of Gold et al. disclosed additional DDD-binding sequences from avariety of AKAP proteins, shown below.

RII-Specific AKAPs AKAP-KL PLEYQAGLLVQNAIQQAI (SEQ ID NO: 82) AKAP79LLIETASSLVKNAIQLSI (SEQ ID NO: 83) AKAP-Lbc LIEEAASRIVDAVIEQVK(SEQ ID NO: 84) RI-Specific AKAPs AKAPce ALYQFADRFSELVISEAL(SEQ ID NO: 85) RIAD LEQVANQLADQIIKEAT (SEQ ID NO: 86) PV38FEELAWKIAKMIWSDVF (SEQ ID NO: 87) Dual-Specificity AKAPs AKAP7ELVRLSKRLVENAVLKAV (SEQ ID NO: 88) MAP2D TAEEVSARIVQVVTAEAV(SEQ ID NO: 89) DAKAP1 QIKQAAFQLISQVILEAT (SEQ ID NO: 90) DAKAP2LAWKIAKMIVSDVMQQ (SEQ ID NO: 91)

Stokka et al. (2006) also developed peptide competitors of AKAP bindingto PKA, shown in SEQ ID NO:92-94. The peptide antagonists weredesignated as Ht31 (SEQ ID NO:92), RIAD (SEQ ID NO:93) and PV-38 (SEQ IDNO:94). The Ht-31 peptide exhibited a greater affinity for the RIIisoform of PKA, while the RIAD and PV-38 showed higher affinity for RI.

Ht31 DLIEEAASRIVDAVIEQVKAAGAY (SEQ ID NO: 92) RIAD LEQYANQLADQIIKEATE(SEQ ID NO: 93) PV-38 FEELAWKIAKMIWSDVFQQC (SEQ ID NO: 94)

Hundsrucker et al. (2006) developed still other peptide competitors forAKAP binding to PKA, with a binding constant as low as 0.4 nM to the DDDof the RII form of PKA. The sequences of various AKAP antagonisticpeptides are provided in Table 1 of Hundsrucker et al., reproduced inTable 10 below. AKAPIS represents a synthetic RII subunit-bindingpeptide. All other peptides are derived from the RII-binding domains ofthe indicated AKAPs.

TABLE 10 AKAP Peptide sequences Peptide Sequence AKAPISQIEYLAKQIVDNAIQQA (SEQ ID NO: 67) AKAPIS-P QIEYLAKQIPDNAIQQA(SEQ ID NO: 95) Ht31 KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 96) Ht31-PKGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 97) AKAP7δ-wt-pepPEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 98) AKAP7δ-L304T-pepPEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 99) AKAP7δ-L308D-pepPEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 100) AKAP7δ-P-pepPEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 101) AKAP7δ-PP-pepPEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 102) AKAP7δ-L314E-pepPEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 103) AKAP1-pepEEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 104) AKAP2-pepLVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 105) AKAP5-pepQYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 106) AKAP9-pepLEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 107) AKAP10-pepNTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 108) AKAP11-pepVNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 109) AKAP12-pepNGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 110) AKAP14-pepTQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 111) Rab32-pepETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO: 112)

Residues that were highly conserved among the AD domains of differentAKAP proteins are indicated below by underlining with reference to theAKAP IS sequence (SEQ ID NO:67). The residues are the same as observedby Alto et al. (2003), with the addition of the C-terminal alanineresidue. (See FIG. 4 of Hundsrucker et al. (2006), incorporated hereinby reference.) The sequences of peptide antagonists with particularlyhigh affinities for the RII DDD sequence were those of AKAP-IS,AKAP7δ-wt-pep, AKAP7δ-L304T-pep and AKAP7δ-L308D-pep.

AKAP-IS QIEYLAKQIVDNAIQQA (SEQ ID NO: 67)

Carr et al. (2001) examined the degree of sequence homology betweendifferent AKAP-binding DDD sequences from human and non-human proteinsand identified residues in the DDD sequences that appeared to be themost highly conserved among different DDD moieties. These are indicatedbelow by underlining with reference to the human PKA RIIα DDD sequenceof SEQ ID NO:65. Residues that were particularly conserved are furtherindicated by italics. The residues overlap with, but are not identicalto those suggested by Kinderman et al. (2006) to be important forbinding to AKAP proteins. The skilled artisan will realize that indesigning sequence variants of DDD, it would be most preferred to avoidchanging the most conserved residues (italicized), and it would bepreferred to also avoid changing the conserved residues (underlined),while conservative amino acid substitutions may be considered forresidues that are neither underlined nor italicized.

(SEQ ID NO: 65) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR LREA R A

The skilled artisan will realize that these and other amino acidsubstitutions in the antibody moiety or linker portions of the DNLconstructs may be utilized to enhance the therapeutic and/orpharmacokinetic properties of the resulting DNL constructs.

Example 17 Generation of TF2 DNL Pretargeting Construct

A trimeric DNL construct designated TF2 was obtained by reactingC-DDD2-Fab-hMN-14 with h679-Fab-AD2. A pilot batch of TF2 was generatedwith >90% yield as follows. Protein L-purified C-DDD2-Fab-hMN-14 (200mg) was mixed with h679-Fab-AD2 (60 mg) at a 1.4:1 molar ratio. Thetotal protein concentration was 1.5 mg/ml in PBS containing 1 mM EDTA.Subsequent steps involved TCEP reduction, HIC chromatography, DMSOoxidation, and IMP 291 affinity chromatography. Before the addition ofTCEP, SE-HPLC did not show any evidence of a₂b formation. Addition of 5mM TCEP rapidly resulted in the formation of a₂b complex consistent witha 157 kDa protein expected for the binary structure. TF2 was purified tonear homogeneity by IMP 291 affinity chromatography (not shown). IMP 291is a synthetic peptide containing the HSG hapten to which the 679 Fabbinds (Rossi et al., 2005, Clin Cancer Res 11:7122s-29s). SE-HPLCanalysis of the IMP 291 unbound fraction demonstrated the removal of a₄,a₂ and free kappa chains from the product (not shown).

Non-reducing SDS-PAGE analysis demonstrated that the majority of TF2exists as a large, covalent structure with a relative mobility near thatof IgG (not shown). The additional bands suggest that disulfideformation is incomplete under the experimental conditions (not shown).Reducing SDS-PAGE shows that any additional bands apparent in thenon-reducing gel are product-related (not shown), as only bandsrepresenting the constituent polypeptides of TF2 were evident (notshown). However, the relative mobilities of each of the fourpolypeptides were too close to be resolved. MALDI-TOF mass spectrometry(not shown) revealed a single peak of 156,434 Da, which is within 99.5%of the calculated mass (157,319 Da) of TF2.

The functionality of TF2 was determined by BIACORE® assay. TF2,C-DDD1-hMN-14+h679-AD1 (used as a control sample of noncovalent a₂bcomplex), or C-DDD2-hMN-14+h679-AD2 (used as a control sample ofunreduced a₂ and b components) were diluted to 1 μg/ml (total protein)and passed over a sensorchip immobilized with HSG. The response for TF2was approximately two-fold that of the two control samples, indicatingthat only the h679-Fab-AD component in the control samples would bind toand remain on the sensorchip. Subsequent injections of WI2 IgG, ananti-idiotype antibody for hMN-14, demonstrated that only TF2 had aDDD-Fab-hMN-14 component that was tightly associated with h679-Fab-AD asindicated by an additional signal response. The additional increase ofresponse units resulting from the binding of WI2 to TF2 immobilized onthe sensorchip corresponded to two fully functional binding sites, eachcontributed by one subunit of C-DDD2-Fab-hMN-14. This was confirmed bythe ability of TF2 to bind two Fab fragments of WI2 (not shown).

Example 18 Production of TF10 Bispecific Antibody for Pretargeting

A similar protocol was used to generate a trimeric TF10 DNL construct,comprising two copies of a C-DDD2-Fab-hPAM4 and one copy ofC-AD2-Fab-679. The cancer-targeting antibody component in TF10 wasderived from hPAM4, a humanized anti-pancreatic cancer mucin MAb thathas been studied in detail as a radiolabeled MAb (e.g., Gold et al.,Clin. Cancer Res. 13: 7380-7387, 2007). The hapten-binding component wasderived from h679, a humanized anti-histaminyl-succinyl-glycine (HSG)MAb. The TF10 bispecific ([hPAM4]₂×h679) antibody was produced using themethod disclosed for production of the (anti CEA)₂× anti HSG bsAb TF2,as described above. The TF10 construct bears two humanized PAM4 Fabs andone humanized 679 Fab.

The two fusion proteins (hPAM4-DDD and h679-AD2) were expressedindependently in stably transfected myeloma cells. The tissue culturesupernatant fluids were combined, resulting in a two-fold molar excessof hPAM4-DDD. The reaction mixture was incubated at room temperature for24 hours under mild reducing conditions using 1 mM reduced glutathione.Following reduction, the DNL reaction was completed by mild oxidationusing 2 mM oxidized glutathione. TF10 was isolated by affinitychromatography using IMP 291-affigel resin, which binds with highspecificity to the h679 Fab.

A full tissue histology and blood cell binding panel has been examinedfor hPAM4 IgG and for an anti-CEA x anti-HSG bsMAb that is enteringclinical trials. hPAM4 binding was restricted to very weak binding tothe urinary bladder and stomach in ⅓ specimens (no binding was seen invivo), and no binding to normal tissues was attributed to the anti-CEA xanti-HSG bsMAb. Furthermore, in vitro studies against cell lines bearingthe H1 and H2 histamine receptors showed no antagonistic or agonisticactivity with the IMP 288 di-HSG peptide, and animal studies in 2different species showed no pharmacologic activity of the peptiderelated to the histamine component at doses 20,000 times higher thanthat used for imaging. Thus, the HSG-histamine derivative does not havepharmacologic activity.

Example 19 Imaging Studies Using Pretargeting With TF10 BispecificAntibody and ¹¹¹In-Labeled Peptides

The following study demonstrates the feasibility of in vivo imagingusing the pretargeting technique with bispecific antibodiesincorporating hPAM4 and labeled peptides. The TF10 bispecific antibody,comprising two copies of a C-DDD2-Fab-hPAM4 and one copy ofC-AD2-Fab-679, was prepared as described in the preceding Example. Nudemice bearing 0.2 to 0.3 g human pancreatic cancer xenografts wereimaged, using pretargeting with TF10 and ¹¹¹In-IMP-288 peptide. Theresults, shown in FIG. 8, demonstrate how clearly delineated tumors canbe detected in animal models using a bsMAb pretargeting method, with¹¹¹In-labeled di-HSG peptide, IMP-288. The six animals in the top ofFIG. 8 received 2 different doses of TF10 (10:1 and 20:1 mole ratio tothe moles of peptide given), and the next day they were given an¹¹¹In-labeled diHSG peptide (IMP 288). The 3 other animals on the bottomof FIG. 8 received only the ¹¹¹In-IMP-288 (no pretargeting). The imageswere taken 3 h after the injection of the labeled peptide and show clearlocalization of 0.2-0.3 g tumors in the pretargeted animals, with nolocalization in the animals given the ¹¹¹In-peptide alone. Tumor uptakeaveraged 20-25% ID/g with tumor/blood ratios exceeding 2000:1,tumor/liver ratios of 170:1, and tumor/kidney ratios of 18/1.

Example 20 Production of Targeting Peptides for Use in Pretargeting and¹⁸F Labeling

In a variety of embodiments, ¹⁸F-labeled proteins or peptides areprepared by a novel technique and used for diagnostic and/or imagingstudies, such as PET imaging. The novel technique for ¹⁸F labelinginvolves preparation of an ¹⁸F-metal complex, preferably an ¹⁸F-aluminumcomplex, which is chelated to a chelating moiety, such as DOTA, NOTA orNETA or derivatives thereof. Chelating moieties may be attached toproteins, peptides or any other molecule using conjugation techniqueswell known in the art. In certain preferred embodiments, the ¹⁸F—Alcomplex is formed in solution first and then attached to a chelatingmoiety that is already conjugated to a protein or peptide. However, inalternative embodiments the aluminum may be first attached to thechelating moiety and the ¹⁸F added later.

Peptide Synthesis

Peptides were synthesized by solid phase peptide synthesis using theFmoc strategy. Groups were added to the side chains of diamino aminoacids by using Fmoc/Aloc protecting groups to allow differentialdeprotection. The Aloe groups were removed by the method of Dangles et.al. (J. Org. Chem. 1987, 52:4984-4993) except that piperidine was addedin a 1:1 ratio to the acetic acid used. The unsymmetrical tetra-t-butylDTPA was made as described in McBride et al. (US Patent Application Pub.No. 2005/0002945, the Examples section of which is incorporated hereinby reference).

The tri-t-butyl DOTA, symmetrical tetra-t-butyl DTPA, ITC-benzyl DTPA,p-SCN-Bn-NOTA and TACN were obtained from MACROCYCLICS® (Dallas, Tex.).The DiBocTACN, NODA-GA(tBu)₃ and the NO₂AtBu were purchased fromCheMatech (Dijon, France). The Aloc/Fmoc Lysine and Dap(diaminopropionic acid derivatives (also Dpr)) were obtained fromCREOSALUS® (Louisville, Ky.) or BACHEM® (Torrance, Calif.). The SieberAmide resin was obtained from NOVABIOCHEM® (San Diego, Calif.). Theremaining Fmoc amino acids were obtained from CREOSALUS®, BACHEM®,PEPTECH® (Burlington, Mass.), EMD BIOSCIENCES® (San Diego, Calif.), CHEMIMPEX® (Wood Dale, Ill.) or NOVABIOCHEM®. The aluminum chloridehexahydrate was purchased from SIGMA-ALDRICH® (Milwaukee, Wis.). Theremaining solvents and reagents were purchased from FISHER SCIENTIFIC®(Pittsburgh, Pa.) or SIGMA-ALDRICH® (Milwaukee, Wis.). ¹⁸F was suppliedby IBA MOLECULAR® (Somerset, N.J.)

¹⁸F-Labeling of IMP 272

The first peptide that was prepared and ¹⁸F-labeled was IMP 272:

DTPA-Gln-Ala-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂MH⁺1512

IMP 272 was synthesized as described (U.S. Pat. No. 7,534,431, theExamples section of which is incorporated herein by reference).

Acetate buffer solution—Acetic acid, 1.509 g was diluted in ˜160 mLwater and the pH was adjusted by the addition of 1 M NaOH then dilutedto 250 mL to make a 0.1 M solution at pH 4.03.

Aluminum acetate buffer solution—A solution of aluminum was prepared bydissolving 0.1028 g of AlCl₃ hexahydrate in 42.6 mL DI water. A 4 mLaliquot of the aluminum solution was mixed with 16 mL of a 0.1 M NaOAcsolution at pH 4 to provide a 2 mM Al stock solution.

IMP 272 acetate buffer solution—Peptide, 0.0011 g, 7.28×10⁻⁷ mol IMP 272was dissolved in 364 μL of the 0.1 M pH 4 acetate buffer solution toobtain a 2 mM stock solution of the peptide.

F-18 Labeling of IMP 272-A 3 μL aliquot of the aluminum stock solutionwas placed in a REACTI-VIAL™ and mixed with 50 μL ¹⁸F (as received) and3 μL of the IMP 272 solution. The solution was heated in a heating blockat 110° C. for 15 min and analyzed by reverse phase HPLC. HPLC analysis(not shown) showed 93% free ¹⁸F and 7% bound to the peptide. Anadditional 10 μL of the IMP 272 solution was added to the reaction andit was heated again and analyzed by reverse phase HPLC (not shown). TheHPLC trace showed 8% ¹⁸F at the void volume and 92% of the activityattached to the peptide. The remainder of the peptide solution wasincubated at room temperature with 150 μL PBS for ˜1 hr and thenexamined by reverse phase HPLC. The HPLC (not shown) showed 58% ¹⁸Funbound and 42% still attached to the peptide. The data indicate that¹⁸F—Al-DTPA complex may be unstable when mixed with phosphate.

The labeled peptide was purified by applying the labeled peptidesolution onto a 1 cc (30 mg) WATERS® HLB column (Part # 186001879) andwashing with 300 μL water to remove unbound F-18. The peptide was elutedby washing the column with 2×100 μL 1:1 EtOH/H₂O. The purified peptidewas incubated in water at 25° C. and analyzed by reverse phase HPLC (notshown). The HPLC analysis showed that the ¹⁸F-labeled IMP 272 was notstable in water. After 40 min incubation in water about 17% of the ¹⁸Fwas released from the peptide, while 83% was retained (not shown).

The peptide (16 μL 2 mM IMP 272, 48 μg) was labeled with ¹⁸F andanalyzed for antibody binding by size exclusion HPLC. The size exclusionHPLC showed that the peptide bound hMN-14×679 but did not bind to theirrelevant bispecific antibody hMN-14×734 (not shown).

IMP 272 ¹⁸F Labeling with Other Metals

A ˜3 μL aliquot of the metal stock solution (6×10⁻⁹ mol) was placed in apolypropylene cone vial and mixed with 75 μL ¹⁸F (as received),incubated at room temperature for ˜2 min and then mixed with 20 μL of a2 mM (4×10⁻⁸ mol) IMP 272 solution in 0.1 M NaOAc pH 4 buffer. Thesolution was heated in a heating block at 100° C. for 15 min andanalyzed by reverse phase HPLC. IMP 272 was labeled with indium (24%),gallium (36%), zirconium (15%), lutetium (37%) and yttrium (2%) (notshown). These results demonstrate that the ¹⁸F metal labeling techniqueis not limited to an aluminum ligand, but can also utilize other metalsas well. With different metal ligands, different chelating moieties maybe utilized to optimize binding of an F-18-metal conjugate.

Production and Use of a Serum-Stable ¹⁸F-Labeled Peptide IMP 449

The peptide, IMP 448 D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ MH⁺1009 wasmade on Sieber Amide resin by adding the following amino acids to theresin in the order shown: Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Aloe wascleaved, Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Aloewas cleaved, Fmoc-D-Ala-OH with final Fmoc cleavage to make the desiredpeptide. The peptide was then cleaved from the resin and purified byHPLC to produce IMP 448, which was then coupled to ITC-benzyl NOTA. Thepeptide, IMP 448, 0.0757 g (7.5×1 mol) was mixed with 0.0509 g(9.09×10⁻⁵ mol) ITC benzyl NOTA and dissolved in 1 mL water. Potassiumcarbonate anhydrous (0.2171 g) was then slowly added to the stirredpeptide/NOTA solution. The reaction solution was pH 10.6 after theaddition of all the carbonate. The reaction was allowed to stir at roomtemperature overnight. The reaction was carefully quenched with 1 M HClafter 14 hr and purified by HPLC to obtain 48 mg of IMP 449.

¹⁸F Labeling of IMP 449

The peptide IMP 449 (0.002 g, 1.37×10⁻⁶ mol) was dissolved in 686 μL (2mM peptide solution) 0.1 M NaOAc pH 4.02. Three microliters of a 2 mMsolution of Al in a pH 4 acetate buffer was mixed with 15 μL, 1.3 mCi of¹⁸F. The solution was then mixed with 20 μL of the 2 mM IMP 449 solutionand heated at 105° C. for 15 min. Reverse Phase HPLC analysis showed 35%(t_(R) ˜10 min) of the activity was attached to the peptide and 65% ofthe activity was eluted at the void volume of the column (3.1 min, notshown) indicating that the majority of activity was not associated withthe peptide. The crude labeled mixture (5 μL) was mixed with pooledhuman serum and incubated at 37° C. An aliquot was removed after 15 minand analyzed by HPLC. The HPLC showed 9.8% of the activity was stillattached to the peptide (down from 35%). Another aliquot was removedafter 1 hr and analyzed by HPLC. The HPLC showed 7.6% of the activitywas still attached to the peptide (down from 35%), which was essentiallythe same as the 15 min trace (data not shown).

High Dose ¹⁸F Labeling

Further studies with purified IMP 449 demonstrated that the ¹⁸F-labeledpeptide was highly stable (91%, not shown) in human serum at 37° C. forat least one hour and was partially stable (76%, not shown) in humanserum at 37° C. for at least four hours. Additional studies wereperformed in which the IMP 449 was prepared in the presence of ascorbicacid as a stabilizing agent. In those studies (not shown), themetal-¹⁸F-peptide complex showed no detectable decomposition in serumafter 4 hr at 37° C. The mouse urine 30 min after injection of¹⁸F-labeled peptide was found to contain ¹⁸F bound to the peptide (notshown). These results demonstrate that the ¹⁸F-labeled peptidesdisclosed herein exhibit sufficient stability under approximated in vivoconditions to be used for ¹⁸F imaging studies.

For studies in the absence of ascorbic acid, ¹⁸F ˜21 mCi in ˜400 μL ofwater was mixed with 9 μL of 2 mM AlCl₃ in 0.1 M pH 4 NaOAc. Thepeptide, IMP 449, 60 μL (0.01 M, 6×10⁻⁷ mol in 0.5 NaOH pH 4.13) wasadded and the solution was heated to 110° C. for 15 min. The crudelabeled peptide was then purified by placing the reaction solution inthe barrel of a 1 cc WATERS® HLB column and eluting with water to removeunbound ¹⁸F followed by 1:1 EtOH/H₂O to elute the ¹⁸F-labeled peptide.The crude reaction solution was pulled through the column into a wastevial and the column was washed with 3×1 mL fractions of water (18.97mCi). The HLB column was then placed on a new vial and eluted with 2×200μL 1:1 EtOH/H₂O to collect the labeled peptide (1.83 mCi). The columnretained 0.1 mCi of activity after all of the elutions were complete. Analiquot of the purified ¹⁸F-labeled peptide (20 μL) was mixed with 200μL of pooled human serum and heated at 37° C. Aliquots were analyzed byreverse phase HPLC. The results showed the relative stability of¹⁸F-labeled purified IMP 449 at 37° C. at time zero, one hour (91%labeled peptide), two hours (77% labeled peptide) and four hours (76%labeled peptide) of incubation in human serum (not shown). It was alsoobserved that ¹⁸F-labeled IMP 449 was stable in TFA solution, which isoccasionally used during reverse phase HPLC chromatography. Thereappears to be a general correlation between stability in TFA andstability in human serum observed for the exemplary ¹⁸F-labeledmolecules described herein. These results demonstrate that ¹⁸F-labeledpeptide, produced according to the methods disclosed herein, showssufficient stability in human serum to be successfully used for in vivolabeling and imaging studies, for example using PET scanning to detectlabeled cells or tissues. Finally, since IMP 449 peptide contains athiourea linkage, which is sensitive to radiolysis, several products areobserved by RP-HPLC. However, when ascorbic acid is added to thereaction mixture, the side products generated were markedly reduced.

Example 21 In Vivo Studies with Pretargeting TF10 DNL Construct and¹⁸F-Labeled Peptide

¹⁸F-labeled IMP 449 was prepared as follows. The ¹⁸F, 54.7 mCi in ˜0.5mL was mixed with 3 μL, 2 mM Al in 0.1 M NaOAc pH 4 buffer. After 3 min10 mL of 0.05 M IMP 449 in 0.5 M pH 4 NaOAc buffer was added and thereaction was heated in a 96° C. heating block for 15 min. The contentsof the reaction were removed with a syringe. The crude labeled peptidewas then purified by HPLC on a C₁₈ column. The flow rate was 3 mL/min.Buffer A was 0.1% TFA in water and Buffer B was 90% acetonitrile inwater with 0.1% TFA. The gradient went from 100% A to 75/25 A:B over 15min. There was about 1 min difference in retention time (t_(R)) betweenthe labeled peptide, which eluted first and the unlabeled peptide. TheHPLC eluent was collected in 0.5 min (mL) fractions. The labeled peptidehad a t_(R) between 6 to 9 min depending on the column used. The HPLCpurified peptide sample was further processed by diluting the fractionsof interest two fold in water and placing the solution in the barrel ofa 1 cc WATERS® HLB column. The cartridge was eluted with 3×1 mL water toremove acetonitrile and TFA followed by 400 μL 1:1 EtOH/H₂O to elute the¹⁸F-labeled peptide. The purified [Al¹⁸F] IMP 449 eluted as a singlepeak on an analytical HPLC C₁₈ column (not shown).

TACONIC® nude mice bearing the four slow-growing sc CaPan1 xenograftswere used for in vivo studies. Three of the mice were injected with TF10(162 μg) followed with [Al¹⁸F] IMP 449 18 h later. TF10 is a humanizedbispecific antibody of use for tumor imaging studies, with divalentbinding to the PAM-4 defined tumor antigen and monovalent binding to HSG(see, e.g., Gold et al., 2007, J. Clin. Oncol. 25(185):4564). One mousewas injected with peptide alone. All of the mice were necropsied at 1 hpost peptide injection. Tissues were counted immediately. Comparison ofmean distributions showed substantially higher levels of ¹⁸F-labeledpeptide localized in the tumor than in any normal tissues in thepresence of tumor-targeting bispecific antibody (data not shown).

Tissue uptake was similar in animals given the [Al¹⁸F] IMP 449 alone orin a pretargeting setting (data not shown). Uptake in the humanpancreatic cancer xenograft, CaPan1, at 1 h was increased 5-fold in thepretargeted animals as compared to the peptide alone (4.6±0.9% ID/g vs.0.89% ID/g). Exceptional tumor/nontumor ratios were achieved at thistime (e.g., tumor/blood and liver ratios were 23.4±2.0 and 23.5±2.8,respectively).

The results demonstrate that ¹⁸F labeled peptide used in conjunctionwith a PAM4 containing antibody construct, such the TF10 DNL construct,provide suitable targeting of the ¹⁸F label to perform in vivo imaging,such as PET imaging analysis.

Example 22 Further Imaging Studies with TF10 Summary

Preclinical and clinical studies have demonstrated the application ofradiolabeled mAb-PAM4 for nuclear imaging and radioimmunotherapy ofpancreatic carcinoma. We have examined herein the ability of the TF10construct to pretarget a radiolabeled peptide for improved imaging andtherapy. Biodistribution studies and nuclear imaging of the radiolabeledTF10 and/or TF10-pretargeted hapten-peptide (IMP-288) were conducted innude mice bearing CaPan1 human pancreatic cancer xenografts. ¹²⁵I-TF10cleared rapidly from the blood, with levels decreasing to <1% injecteddose per gram (ID/g) by 16 hours. Tumor uptake was 3.47±0.66% ID/g atthis time point with no accumulation in any normal tissue. To show theutility of the pretargeting approach, ¹¹¹In-IMP-288 was administered 16hours after TF10. At 3 hours postadministration of radiolabeled peptide,imaging showed intense uptake within the tumors and no evidence ofaccretion in any normal tissue. No targeting was observed in animalsgiven only the ¹¹¹In-peptide. Tumor uptake of the TF10-pretargeted¹¹¹In-IMP-288 was 24.3±1.7% ID/g, whereas for ¹¹¹In-IMP-288 alone it wasonly 0.12±0.002% ID/g at 16 hours. Tumor/blood ratios were significantlygreater for the pretargeting group(˜1,000:1 at 3 hours) compared with¹¹¹In-PAM4-IgG (˜5:1 at 24 hours; P<0.0003). Radiation dose estimatessuggested that TF10/⁹⁰Y-peptide pretargeting would provide a greaterantitumor effect than ⁹⁰Y-PAM4-IgG. Thus, the results support that TF10pretargeting may provide improved imaging for early detection,diagnosis, and treatment of pancreatic cancer as compared with directlyradiolabeled PAM4-IgG. (Gold et al., Cancer Res 2008, 68(12):4819-26)

We have identified a unique biomarker present on mucin expressed by >85%of invasive pancreatic adenocarcinomas, including early stage I diseaseand the precursor lesions, pancreatic intraepithelial neoplasia andintraductal papillary mucinous neoplasia (Gold et al., Clin Cancer Res2007, 13:7380-87). The specific epitope, as detected by mAb-PAM4 (Goldet al., Int J Cancer 1994, 57:204-10), is absent from normal andinflamed pancreatic tissues, as well as most other malignant tissues.Thus, detection of the epitope provides a high diagnostic likelihood forthe presence of pancreatic neoplasia. Early clinical studies using ¹³¹I-and ^(94m)Tc-labeled, murine PAM4 IgG or Fab′, respectively, showedspecific targeting in 8 of 10 patients with invasive pancreaticadenocarcinoma (Mariani et al., Cancer Res 1995, 55:5911s-15s; Gold etal., Crit. Rev Oncol Hematol 2001, 39:147-54). Of the two negativepatients, one had a poorly differentiated pancreatic carcinoma that didnot express the PAM4-epitope, whereas the other patient was later foundto have pancreatitis rather than a malignant lesion.

Accordingly, the high specificity of PAM4 for pancreatic cancer is ofuse for the detection and diagnosis of early disease. In addition toimproved detection, ⁹⁰Y-PAM4 IgG was found to be effective in treatinglarge human pancreatic cancer xenografts in nude mice (Cardillo et al.,Clin Cancer Res 2001, 7:3186-92), and when combined with gemcitabine,further improvements in therapeutic response were observed (Gold et al.,Clin Cancer Res 2004, 10:3552-61; Gold et al., Int J Cancer 2004,109:618-26). A Phase I therapy trial in patients who failed gemcitabinetreatment was recently completed, finding the maximum tolerated dose of⁹⁰Y-humanized PAM4 IgG to be 20 mCi/m² (Gulec et al., Proc Amer Soc ClinOne, 43rd Annual Meeting, J Clin Oncol 2007, 25(18S):636s). Although allpatients showed disease progression at or after week 8, initialshrinkage of tumor was observed in several cases. Clinical studies arenow underway to evaluate a fractionated dosing regimen of ⁹⁰Y-hPAM4 IgGin combination with a radiosensitizing dose of gemcitabine.

We report herein the development of a novel recombinant, humanizedbispecific monoclonal antibody (mAb), TF10, based on the targetingspecificity of PAM4 to pancreatic cancer. This construct also binds tothe unique synthetic hapten, histamine-succinyl-glycine (HSG), which hasbeen incorporated in a number of small peptides that can be radiolabeledwith a wide range of radionuclides suitable for single-photon emissioncomputed tomography (SPECT) and positron emission tomography (PET)imaging, as well as for therapeutic purposes (Karacay et al., ClinCancer Res 2005, 11:7879-85; Sharkey et al., Leukemia 2005, 19:1064-9;Rossi et al., Proc Natl Acad Sci USA 2006, 103:6841-6; McBride et al., JNucl Med 2006, 47:1678-88). These studies illustrate the potential ofthis new construct to target pancreatic adenocarcinoma for imaging ortherapeutic applications.

Methods and Materials

The TF2 and TF10 bispecific DNL constructs and the IMP 288 targetingpeptide were prepared as described above. Sodium iodide (¹²⁵I) andindium chloride (¹¹¹In) were obtained from PERKIN-ELMER®. TF10 wasroutinely labeled with ¹²⁵I by the iodogen method, with purification byuse of size-exclusion spin columns. Radiolabeling of DOTA-peptide andDOTA-PAM4-IgG with ¹¹¹InCl was done as previously described (Rossi etal., Proc Natl Acad Sci USA 2006, 103:6841-6; McBride et al., J Nucl Med2006, 47:1678-88). Purity of the radiolabeled products was examined bysize-exclusion high-performance liquid chromatography with the amount offree, unbound isotope determined by instant TLC.

For TF10 distribution studies, female athymic nude mice ˜20 g (TACONIC®Farms), bearing s.c. CaPan1 human pancreatic cancer xenografts, wereinjected with ¹²⁵I-TF10 (10 μCi; 40 μg, 2.50×10⁻¹⁰ mol). At various timepoints, groups of mice (n=5) were necropsied, with tumor and nontumortissues removed and counted in a gamma counter to determine thepercentage of injected dose per gram of tissue (% ID/g), with thesevalues used to calculate blood clearance rates and tumor/nontumorratios.

For pretargeting biodistribution studies, a bispecific mAb/radiolabeledpeptide molar ratio of 10:1 was used. For example, a group of athymicnude mice bearing s.c. CaPan1 human pancreatic cancer xenografts wasadministered TF10 (80 μg, 5.07×10⁻¹⁰ mol), whereas a second group wasleft untreated. At 16 h postinjection of TF10, ¹¹1n-IMP-288hapten-peptide (30 μCi, 5.07×10⁻¹¹ mol) was administered. Mice werenecropsied at several time points, with tumor and nontumor tissuesremoved and counted in a gamma counter to determine the % ID/g.Tumor/nontumor ratios were calculated from these data. In a separatestudy, groups of mice were given ¹¹¹In-DOTA-PAM4-IgG (20 μC, 50 μg,3.13×10⁻¹⁰ mol) for the purpose of comparing biodistribution, nuclearimaging, and potential therapeutic activity. Radiation dose estimateswere calculated from the time-activity curves with the assumption of noactivity at zero time. Student's t test was used to assess significantdifferences.

To perform nuclear immunoscintigraphy, at 3 h postinjection ofradiolabeled peptide or 24 h postinjection of radiolabeled hPAM4-IgG,tumor-bearing mice were imaged with a dual-head Solus gamma camerafitted with medium energy collimator for ¹¹¹In (ADAC Laboratories). Micewere imaged for a total of 100,000 cpm or 10 min, whichever came first.

Results

In vitro characterization of the bispecific mAb TF10. The binding ofTF10 to the target mucin antigen was analyzed by ELISA (FIG. 9). Theresults showed nearly identical binding curves for the divalent TF10,PAM4-IgG, and PAM4-F(ab′)₂ (half-maximal binding was calculated as1.42±0.10, 1.31±0.12, and 1.83±0.16 nmol/L, respectively; P>0.05 forall), whereas the monovalent bsPAM4 chemical conjugate (PAM4-Fab′ xanti-DTPA-Fab′) had a significantly lower avidity (half-maximal binding,30.61±2.05 nmol/L; P=0.0379, compared with TF10), suggesting that TF10binds in a divalent manner. The immunoreactive fraction of ¹²⁵I-TF10bound to the mucin was 87%, with 9% found as unbound TF10 and 3% as freeiodide (not shown). Ninety percent of the ¹¹¹In-IMP-288 bound to TF10(not shown). Of the total ¹¹¹In-IMP-288 bound to TF10, 92% eluted athigher molecular weight when excess mucin (200 μg) was added, with only3% eluting with the non-mucin-reactive TF10 fraction. An additional 5%of the radiolabeled peptide eluted in the free peptide volume. None ofthe radiolabeled peptide bound to the mucin antigen in the absence ofTF10 (not shown).

Biodistribution of ¹²⁵I-TF10 in CaPan1 tumor-bearing nude mice. TF10showed a rapid clearance from the blood, starting with 21.03±1.93% ID/gat 1 hour and decreasing to just 0.13±0.02% ID/g at 16 hours. Thebiological half-life was calculated to be 2.19 hours [95% confidenceinterval (95% CI), 2.11-2.27 hours]. Tissue uptake revealed enhancedactivity in the liver, spleen, and kidneys at 1 hour, which cleared justas quickly by 16 hours [T_(1/2)=2.09 hours (95% CI, 2.08-2.10), 2.84hours (95% CI, 2.49-3.29), and 2.44 hours (95% CI, 2.28-2.63) for liver,spleen, and kidney, respectively]. Activity in the stomach most likelyreflects the accretion and excretion of radioiodine, suggesting that theradioiodinated TF10 was actively catabolized, presumably in the liverand spleen, thereby explaining its rapid clearance from the blood.Nevertheless, by 16 hours, the concentration of radioiodine within thestomach was below 1% ID/g. A group of five non-tumor-bearing nude micegiven ¹²⁵I-TF10 and necropsied at 16 hours showed similar tissuedistribution, suggesting that the tumor had not affected the bispecificmAb distribution and clearance from normal tissues (data not shown). Ofcourse, it is possible that differences occurred before the initial timepoint examined. Tumor uptake of TF10 peaked at 6 hours postinjection(7.16±1.10% ID/g) and had decreased to half maximum binding (3.47±0.66%ID/g) at 16 hours. Tumor uptake again decreased nearly 2-fold over thenext 32 hours, but then was stable over the following 24 hours.

Biodistribution of TF10-pretargeted, ¹¹¹In-labeled peptide. Althoughmaximum tumor uptake of TF10 occurred at 6 hours, previous experienceindicated that the radiolabeled peptide would need to be given at a timepoint when blood levels of TF10 had cleared to <1% ID/g (i.e., 16hours). Higher levels of TF10 in the blood would lead to unacceptablyhigh binding of the radiolabeled peptide within the blood (i.e., lowtumor/blood ratios), whereas administering the peptide at a later timewould mean the concentration of TF10 in the tumor would be decreasedwith consequently reduced concentration of radiolabeled peptide withinthe tumor. Thus, an initial pretargeting study was done using a 16-hourinterval. With the amount of the ¹¹¹In-IMP-288 held constant (30 μCi,5.07×10⁻¹¹ mol), increasing amounts of TF10 were given so that theadministered dose of TF10 and IMP-288 expressed as mole ratio variedfrom 5:1 to 20:1 (Table 11).

TABLE 11 Biodistribution of ¹¹¹In-IMP-288 alone (no TF10) or pretargetedwith varying amounts of TF10 % ID/g at 3 h (mean ± SD) 5:1 10:1 20:1 NoTF10 Tumor 19.0 ± 3.49 24.3 ± 1.71 28.6 ± 0.73 0.12 ± 0.00 Liver 0.09 ±0.01 0.21 ± 0.12 0.17 ± 0.01 0.07 ± 0.00 Spleen 0.12 ± 0.04 0.16 ± 0.070.26 ± 0.10 0.04 ± 0.01 Kidneys 1.59 ± 0.11 1.72 ± 0.24 1.53 ± 0.14 1.71± 0.22 Lungs 0.19 ± 0.06 0.26 ± 0.00 0.29 ± 0.04 0.03 ± 0.00 Blood 0.01± 0.00 0.01 ± 0.01 0.01 ± 0.00 0.00 ± 0.00 Stomach 0.03 ± 0.02 0.02 ±0.02 0.01 ± 0.00 0.02 ± 0.01 Small intestine 0.12 ± 0.08 0.08 ± 0.030.04 ± 0.01 0.06 ± 0.02 Large intestine 0.23 ± 0.10 0.39 ± 0.08 0.25 ±0.08 0.33 ± 0.02 Pancreas 0.02 ± 0.00 0.02 ± 0.01 0.02 ± 0.00 0.02 ±0.00 Tumor weight 0.12 ± 0.03 0.32 ± 0.09 0.27 ± 0.01 0.35 ± 0.03 (g)

At 3 hours the amount of ¹¹¹In-IMP-288 in the blood was barelydetectable (0.01%). Tumor uptake increased from 19.0±3.49% ID/g to28.55±0.73% ID/g as the amount of bispecific mAb administered wasincreased 4-fold (statistically significant differences were observedfor comparison of each TF10/peptide ratio, one group to another; P<0.03or better), but without any appreciable increase in normal tissueuptake. Tumor uptake in the animals given TF10 was >100-fold higher thanwhen ¹¹¹In-IMP-288 was given alone. Comparison of ¹¹¹In activity in thenormal tissues of the animals that either received or did not receiveprior administration of TF10 indicated similar absolute values, which inmost instances were not significantly different. This suggests that thebispecific mAb had cleared sufficiently from all normal tissues by 16hours to avoid appreciable peptide uptake in these tissues. Tumor/bloodratios were >2,000:1, with other tissue ratios exceeding 100:1. Eventumor/kidney ratios exceeded 10:1. The highest tumor uptake ofradioisotope with minimal targeting to nontumor tissues resulted fromthe 20:1 ratio; however, either of the TF10/peptide ratios could be usedto achieve exceptional targeting to tumor, both in terms of signalintensity and contrast ratios. The 10:1 ratio was chosen for furtherstudy because the absolute difference in tumor uptake of radiolabeledpeptide was not substantially different between the 10:1 (24.3±1.71%ID/g) and 20:1 (28.6±0.73% ID/g) ratios.

Images of the animals given TF10-pretargeted ¹¹¹In-IMP-288 at abispecific mAb/peptide ratio of 10:1, or the ¹¹¹In-IMP-288 peptidealone, are shown in FIG. 10. The majority of these tumors were 1.0.5 cmin diameter, weighing ˜0.25 g. The images show highly intense uptake inthe tumor of the TF10-pretargeted animals (FIG. 10A). The intensity ofthe image background for the TF10-pretargeted animals was increased tomatch the intensity of the image taken of the animals given the¹¹¹In-IMP-288 alone (FIG. 10B). However, when the images were optimizedfor the TF10-pretargeted mice, the signal intensity and contrast were sohigh that no additional activity was observed in the body. No tumorlocalization was seen in the animals given the ¹¹¹In-IMP-288 alone, evenwhen image intensity was enhanced (FIG. 10C).

An additional experiment was done to assess the kinetics of targeting¹¹¹In-hPAM4 whole-IgG compared with that of the TF10-pretargeted¹¹¹In-IMP-288 peptide. Tumor uptake of the ¹¹¹In-peptide was highest atthe initial time point examined, 3 hours (15.99±4.11% ID/g), whereas theblood concentration of radiolabeled peptide was only 0.02±0.01% ID/g,providing a mean tumor/blood ratio of 946.3±383.0. Over time,radiolabeled peptide cleared from the tumor with a biological half-lifeof 76.04 hours. Among nontumor tissues, uptake was highest in thekidneys, averaging 1.89±0.42% ID/g at 3 hours with a steady decreaseover time (biological half-life, 33.6 hours). Liver uptake started at0.15±0.06% ID/g and remained essentially unchanged over time. Incontrast to the TF10-pretargeted ¹¹¹In-IMP-288, the ¹¹¹In-hPAM4-IgG hada slower clearance from the blood, albeit there was a substantialclearance within the first 24 hours, decreasing from 30.1% ID/g at 3hours to just 11.5±1.7% ID/g at 24 hours. Variable elevated uptake inthe spleen suggested that the antibody was likely being removed from theblood by targeting of secreted mucin that had become entrapped withinthe spleen. Tumor uptake peaked at 48 hours with 80.4±6.1% ID/g, andremained at an elevated level over the duration of the monitoringperiod. The high tumor uptake, paired with a more rapid than expectedblood clearance for an IgG, produced tumor/blood ratios of 5.2±1.0within 24 hours. FIG. 10C shows the images of the animals at 24 hourspostadministration of ¹¹¹In-PAM4-IgG, illustrating that tumors could bevisualized at this early time, but there was still considerable activitywithin the abdomen. Tumor/nontumor ratios were mostly higher forTF10-pretargeted ¹¹¹In-labeled hapten-peptide as compared with¹¹¹In-hPAM4-IgG, except for the kidneys, where tumor/kidney ratios withthe ¹¹¹In-IMP-288 and ¹¹¹In-hPAM4-IgG were similar at later times.However, tumor/kidney ratios for the TF10-pretargeted ¹¹¹In-IMP-288 werehigh enough (e.g., ˜7:1) at 3 hours to easily discern tumor from normaltissue.

FIG. 11 illustrates the potential therapeutic capability of the directand pretargeted methods to deliver radionuclide (⁹⁰Y). Although theconcentration (% ID/g) of radioisotope within the tumor seems to be muchgreater when delivered by PAM4-IgG than by pretargeted TF10 at theirrespective maximum tolerated dose (0.15 mCi for ⁹⁰Y-hPAM4 and 0.9 mCifor TF10-pretargeted ⁹⁰Y-IMP-288), the radiation dose to tumor would besimilar (10,080 and 9,229 cGy for ⁹⁰Y-PAM4-IgG and TF10-pretargeted⁹⁰Y-IMP-288, respectively). The advantage for the pretargeting methodwould be the exceptionally low activity in blood (9 cGy), almost200-fold less than with the ⁹⁰Y-hPAM4 IgG (1,623 cGy). It is alsoimportant to note that the radiation dose to liver, as well as othernontumor organs, would be much lower with the TF10-pretargeted⁹⁰Y-IMP-288. The exception would be the kidneys, where the radiationdose would be similar for both protocols at their respective maximumdose (612 and 784 cGy for ⁹⁰Y-PAM4-IgG and TF10-⁹⁰Y-IMP-288,respectively). The data suggest that for ⁹⁰Y-PAM4-IgG, as with mostother radiolabeled whole-IgG mAbs, the dose-limiting toxicity would behematologic; however, for the TF10 pretargeting protocol, thedose-limiting toxicity would be the kidneys.

Discussion

Current diagnostic modalities such as ultrasound, computerizedtomography (CT), and magnetic resonance imaging (MRI) technologies,which provide anatomic images, along with PET imaging of the metabolicenvironment, have routinely been found to provide high sensitivity inthe detection of pancreatic masses. However, these data are, for themost part, based on detection of lesions >2 cm in a population that isalready presenting clinical symptoms. At this time in the progression ofthe pancreatic carcinoma, the prognosis is rather dismal. To improvepatient outcomes, detection of small, early pancreatic neoplasms in theasymptomatic patient is necessary.

Imaging with a mAb-targeted approach, such as is described herein withmAb-PAM4, may provide for the diagnosis of these small, early cancers.Of prime importance is the specificity of the mAb. We have presentedconsiderable data, including immunohistochemical studies of tissuespecimens (Gold et al., Clin Cancer Res 2007; 13:7380-7; Gold et al.,Int J Cancer 1994; 57:204-10) and immunoassay of patient sera (Gold etal., J Clin Oncol 2006; 24:252-8), to show that mAb-PAM4 is highlyreactive with a biomarker, the presence of which provides highdiagnostic likelihood of pancreatic neoplasia. Furthermore, wedetermined that PAM4, although not reactive with normal adult pancreatictissues nor active pancreatitis, is reactive with the earliest stages ofneoplastic progression within the pancreas (pancreatic intraepithelialneoplasia 1 and intraductal papillary mucinous neoplasia) and that thebiomarker remains at high levels of expression throughout theprogression to invasive pancreatic adenocarcinoma (Gold et al., ClinCancer Res 2007; 13:7380-7). Preclinical studies with athymic nude micebearing human pancreatic tumor xenografts have shown specific targetingof radiolabeled murine, chimeric, and humanized versions of PAM4.

In the current studies, we have examined a next-generation, recombinant,bispecific PAM4-based construct, TF10, which is divalent for the PAM4arm and monovalent for the anti-HSG hapten arm. There are severalimportant characteristics of this pretargeting system's constructs,named dock-and-lock, including its general applicability and ease ofsynthesis. However, for the present consideration, the major differencesfrom the previously reported chemical construct are the valency, whichprovides improved binding to tumor antigen, and, importantly, itspharmacokinetics. TF10 clearance from nontumor tissues is much morerapid than was observed for the chemical conjugate. Time for bloodlevels of the bispecific constructs to reach less than 1% ID/g was 40hours postinjection for the chemical construct versus 16 hours for TF10.A more rapid clearance of the pretargeting agent has provided a vastimprovement of the tumor/blood ratio, while maintaining high signalstrength at the tumor site (% ID/g).

In addition to providing a means for early detection and diagnosis, theresults support the use of the TF10 pretargeting system for cancertherapy. Consideration of the effective radiation dose to tumor andnontumor tissues favors the pretargeting method over directlyradiolabeled PAM4-IgG. The dose estimates suggest that the two deliverysystems have different dose-limiting toxicities: myelotoxicity for thedirectly radiolabeled PAM4 versus the kidney for the TF10 pretargetingsystem. This is of significance for the future clinical development ofradiolabeled PAM4 as a therapeutic agent.

Gemcitabine, the frontline drug of choice for pancreatic cancer, canprovide significant radiosensitization of tumor cells. In previousstudies, we showed that combinations of gemcitabine and directlyradiolabeled PAM4-IgG provided synergistic antitumor effects comparedwith either arm alone (Gold et al., Clin Cancer Res 2004, 10:3552-61;Gold et al., Int J Cancer 2004, 109:618-26). The dose-limiting factorwith this combination was overlapping hematologic toxicity. However,because the dose-limiting organ for TF10 pretargeting seems to be thekidney rather than hematologic tissues, combinations with gemcitabineshould be less toxic, thus allowing increased administration ofradioisotope with consequently greater antitumor efficacy.

The superior imaging achieved with TF10 pretargeting in preclinicalmodels, as compared with directly radiolabeled DOTA-PAM4-IgG, provides acompelling rationale to proceed to clinical trials with this imagingsystem. The specificity of the tumor-targeting mAb for pancreaticneoplasms, coupled with the bispecific antibody platform technologyproviding the ability to conjugate various imaging compounds to theHSG-hapten-peptide for SPECT (¹¹¹In), PET (⁶⁸Ga), ultrasound (Au), orother contrast agents, or for that matter ⁹⁰Y or other radionuclides fortherapy, provides high potential to improve overall patient outcomes(Goldenberg et al., J Nucl Med 2008, 49:158-63). In particular, webelieve that a TF10-based ImmunoPET procedure will have major clinicalvalue to screen individuals at high-risk for development of pancreaticcancer (e.g., genetic predisposition, chronic pancreatitis, smokers,etc.), as well as a means for follow-up of patients with suspiciousabdominal images from conventional technologies and/or with indicationsdue to the presence of specific biomarker(s) or abnormal biochemicalfindings. When used as part of an ongoing medical plan for followingthese patients, early detection of pancreatic cancer may be achieved.Finally, in combination with gemcitabine, TF10 pretargeting may providea better opportunity for control of tumor growth than directlyradiolabeled PAM4-IgG.

Example 23 Therapy of Pancreatic Cancer Xenografts with Gemcitabine and⁹⁰Y-Labeled Peptide Pretargeted Using TF10

Summary

⁹⁰Y-hPAM4 IgG is currently being examined in Phase I/II trials incombination with gemcitabine in patients with Stage III/IV pancreaticcancer. We disclose a new approach for pretargeting radionuclides thatis able to deliver a similar amount of radioactivity to pancreaticcancer xenografts, but with less hematological toxicity, which would bemore amenable for combination with gemcitabine. Nude mice bearing ˜0.4cm³ sc CaPan1 human pancreatic cancer were administered a recombinantbsMAb, TF10, followed 1 day later with a ⁹⁰Y-labeled hapten-peptide(IMP-288). Various doses and schedules of gemcitabine were added to thistreatment, and tumor progression monitored up to 28 weeks. 0.7 mCiPT-RAIT alone produce only a transient 60% loss in blood counts, andanimals given 0.9 mCi of PT-RAIT alone and 0.7 mCi PT-RAIT+6 mggemcitabine (human equivalent ˜1000 mg/m²) had no histological evidenceof renal toxicity after 9 months. A single dose of 0.25 or 0.5 mCiPT-RAIT alone can completely ablate 20% and 80% of the tumors,respectively. Monthly fractionated PT-RAIT (0.25 mCi/dose given at thestart of each gemcitabine cycle) added to a standard gemcitabine regimen(6 mg wkly x 3; 1 wk off; repeat 3 times) significantly increased themedian time for tumors to reach 3.0 cm³ over PT-RAIT alone. Othertreatment plans examining non-cytotoxic radiosensitizing dose regimensof gemcitabine added to PT-RAIT also showed significant improvements intreatment response over PT-RAIT alone. The results show that PT-RAIT isa promising new approach for treating pancreatic cancer. Current dataindicate combining PT-RAIT with gemcitabine will enhance therapeuticresponses.

Methods

TF10 bispecific antibody was prepared as described above. Forpretargeting, TF10 was given to nude mice bearing the human pancreaticadenocarcinoma cell line, CaPan1. After allowing sufficient time forTF10 to clear from the blood (16h), the radiolabeled divalentHSG-peptide was administered. The small molecular weight HSG-peptide(−1.4 kD) clears within minutes from the blood, entering theextravascular space where it can bind to anti-HSG arm of the pretargetedTF10 bsMAb. Within a few hours, >80% of the radiolabeled HSG-peptide isexcreted in the urine, leaving the tumor localized peptide and a traceamount in the normal tissues.

Results

FIG. 12 illustrates the therapeutic activity derived from a singletreatment of established (˜0.4 cm³) CaPan1 tumors with 0.15 mCi of⁹⁰Y-hPAM4 IgG, or 0.25 or 0.50 mCi of TF10-pretargeted ⁹⁰Y-IMP-288.Similar anti-tumor activity was observed for the 0.5-mCi pretargeteddose vs. 0.15-mCi dose of the directly radiolabeled IgG, buthematological toxicity was severe at this level of the direct conjugate(not shown), while the pretargeted dose was only moderately toxic (notshown). Indeed, the MTD for pretargeting using 90Y-IMP-288 is at least0.9 mCi in nude mice.

FIG. 13 shows that the combination of gemcitabine and PT-BAIT has asynergistic effect on anti-tumor therapy. Human equivalent doses of 1000mg/m² (6 mg) of gemcitabine (GEM) were given intraperitoneally to miceweekly for 3 weeks, then after resting for 1 week, this regimen wasrepeated 2 twice. PT-RAIT (0.25 mCi of TF10-pretargeted ⁹⁰Y-IMP-288) wasgiven 1 day after the first GEM dose in each of the 3 cycles oftreatment. Gem alone had no significant impact on tumor progression(survival based on time to progress to 3.0 cm³). PT-RAIT alone improvedsurvival compared to untreated animals, but the combined GEM withPT-RAIT regimen increased the median survival by nearly 10 weeks.Because hematological toxicity is NOT dose-limiting for PT-RAIT, but itis one of the limitations for gemcitabine therapy, these studies suggestthat PT-RAIT could be added to a standard GEM therapy with the potentialfor enhanced responses. The significant synergistic effect ofgemcitabine plus PT-RAIT was surprising and unexpected.

A further study examined the effect of the timing of administration onthe potentiation of anti-tumor effect of gemcitabine plus PT-RAIT. Asingle 6-mg dose of GEM was given one day before or 1 day after 0.25 mCiof TF10-pretargeted ⁹⁰Y-IMP-288 (not shown). This study confirmed whatis already well known with GEM, i.e., radiosensitization is best givenin advance of the radiation. Percent survival of treated mice showedlittle difference in survival time between PT-RAIT alone and PT-RAITwith gemcitabine given 22 hours after the radiolabeled peptide. However,administration of gemcitabine 19 hours prior to PT-RAIT resulted in asubstantial increase in survival (not shown).

Single PT-RAIT (0.25 mCi) combined with cetuximab (1 mg weekly ip; 7weeks) or with cetuximab+GEM (6 mg weekly×3) in animals bearing CaPan1showed the GEM+cetuximab combination with PT-RAIT providing a betterinitial response (FIG. 14), but the response associated with justcetuximab alone added to PT-RAIT was encouraging (FIG. 14), since it wasas good or better than PT-RAIT+GEM. Because the overall survival in thisstudy was excellent, with only 2 tumors in each group progressingto >2.0 cm³ after 24 weeks when the study was terminated, these resultsindicate a potential role for cetuximab when added to PT-RAIT.

Example 24 Effect of Fractionated Pretargeted Radioimmunotherapy(PT-RAIT) for Pancreatic Cancer Therapy

We evaluated fractionated therapy with ⁹° Y-DOTA-di-HSG peptide(IMP-288) and TF10. Studies using TF10 and radiolabeled IMP-288 wereperformed in nude mice bearing s.c. CaPan1 human pancreatic cancerxenografts, 0.32-0.54 cm³. For therapy, TF10-pretargeted ⁹⁰Y-IMP-288 wasgiven [A] once (0.6mCi on wk 0) or [B] fractionated (0.3 mCi on wks 0and 1), [C] (0.2 mCi on wks 0, 1 and 2) or [D] (0.2 mCi on wks 0, 1 and4).

Tumor regression (>90%) was observed in the majority of mice, 9/10,10/10, 9/10 and 8/10 in groups [A], [B], [C] and [D], respectively. Ingroup [A], maximum tumor regression in 50% of the mice was reached at3.7 wks, compared to 6.1, 8.1 and 7.1 wks in [B], [C] and [D],respectively. Some tumors showed regrowth. At week 14, the besttherapeutic response was observed in the fractionated group (2×0.3 mCi),with 6/10 mice having no tumors (NT) compared to 3/10 in the 3×0.2 mCigroups and 1/10 in the 1×0.6mCi group. No major body weight loss wasobserved. Fractionated PT-RAIT provides another alternative for treatingpancreatic cancer with minimum toxicity.

Example 25 ⁹⁰Y-hPAM4 Radioimmunotherapy (RAIT) Plus RadiosensitizingGemcitabine (GEM) Treatment in Advanced Pancreatic Cancer (PC)

⁹⁰Y-hPAM4, a humanized antibody highly specific for PC, showed transientactivity in patients with advanced disease, and GEM enhanced RAIT inpreclinical studies. This study evaluated repeated treatment cycles of⁹⁰Y-hPAM4 plus GEM in patients with untreated, unresectable PC. The⁹⁰Y-dose was escalated by cohort, with patients repeating 4-wk cycles(once weekly 200 mg/m² GEM, ⁹⁰Y-hPAM4 once-weekly wks 2-4) untilprogression or unacceptable toxicity. Response assessments used CT,FDG-PET, and CA19.9 serum levels.

Of 8 patients (3F/5M, 56-72 y.o.) at the 1^(st) 2 dose levels (6.5 and9.0 mCi/m² ⁹⁰Y-IRAM4×3), hematologic toxicity has been transient Grade1-2. Two patients responded to initial treatment with FDG SUV and CA19.9decreases, and lesion regression by CT. Both patients continue in goodperformance status now after 9 and 11 mo. and after a total of 3 and 4cycles, respectively, without additional toxicity. A 3^(rd) patient witha stable response by PET and CT and decreases in CA19.9 levels afterinitial treatment is now undergoing a 2nd cycle. Four other patients hadearly disease progression and the remaining patient is still beingevaluated. Dose escalation is continuing after fractionated RAIT with⁹⁰Y-hPAM4 plus low-dose gemcitabine demonstrated therapeutic activity atthe initial ⁹⁰Y-dose levels, with minimal hematologic toxicity, evenafter 4 therapy cycles.

Example 26 Early Detection of Pancreatic Carcinoma Using Mab-PAM4 and InVitro Immunoassay

Immunohistochemistry studies were performed with PAM4 antibody. Resultsobtained with stained tissue sections showed no reaction of PAM4 withnormal pancreatic ducts, ductules and acinar tissues (not shown). Incontrast, use of the MA5 antibody applied to the same tissue samplesshowed diffuse positive staining of normal pancreatic ducts and acinartissue (not shown). In tissue sections with well differentiated ormoderately differentiated pancreatic adenocarcinoma, PAM4 staining waspositive, with mostly cytoplasmic staining but intensification of at thecell surface. Normal pancreatic tissue in the same tissue sections wasunstained.

Table 12 shows the results of immunohistochemical analysis with PAM4 MAbin pancreatic adenocarcinoma samples of various stages ofdifferentiation. Overall, there was an 87% detection rate for allpancreatic cancer samples, with 100% detection of well differentiatedand almost 90% detection of moderately differentiated pancreaticcancers.

TABLE 12 PAM4 Labeling Pattern Cancer n Focal Diffuse Total Well Diff.13 2 11  13 (100%) Moderately Diff. 24 6 15 21 (88%) Poorly Diff. 18 5 914 (78%) Total 55 13 35 48 (87%)

Table 13 shows that PAM4 immunohistochemical staining also detected avery high percentage of precursor lesions of pancreatic cancer,including PanIn-1A to PanIN-3, IPMN (intraductal papillary mucinousneoplasms) and MCN (mucinous cystic neoplasms). Overall, PAM4 stainingdetected 89% of all pancreatic precursor lesions. These resultsdemonstrate that PAM4 antibody-based immunodetection is capable ofdetecting almost 90% of pancreatic cancers and precursor lesions by invitro analysis. PAM4 expression was observed in the earliest phases ofPanIN development. Intense staining was observed in IPMN and MCN samples(not shown). The PAM4 epitope was present at high concentrations(intense diffuse stain) in the great majority of pancreaticadenocarcinomas. PAM4 showed diffuse, intense reactivity with theearliest stages of pancreatic carcinoma precursor lesions, includingPanIN-1, IPMN and MCN, yet was non-reactive with normal pancreatictissue. Taken together, these results show that diagnosis and/ordetection with the PAM4 antibody is capable of detecting, with very highspecificity, the earliest stages of pancreatic cancer development.

TABLE 13 PAM4 Labeling Pattern n Focal Diffuse Total PanIn-1A 27 9 15 24(89%) PanIn-1B 20 4 16  20 (100%) PanIn-2 11 6 4 10 (91%) PanIn-3 5 2 0 2 (40%) Total PanIn 63 21 35 56 (89%) IPMN 36 6 25 31 (86%) MCN 27 3 2225 (92%)

An enzyme based immunoassay for PAM4 antigen in serum samples wasdeveloped. FIG. 15 shows the results of differential diagnosis usingPAM4 immunoassay for pancreatic cancer versus normal tissues and othertypes of cancer. The results showed a sensitivity of detection ofpancreatic cancer of 77.4%, with a specificity of detection of 94.3%,comparing pancreatic carcinoma (n=53) with all other specimens (n=233),including pancreatitis and breast, ovarian and colorectal cancer andlymphoma. The data of FIG. 15 are presented in tabular form in Table 14.

TABLE 14 PAM4-Reactive Mucin in Patient Sera n Mean SD Median Range #Positive (%) Normal 43 0.1 0.3 0.0 0-2.0  0 (0) Pancreatitis 87 3.0 11.50.0 0-63.6 4 (5) Pancreatic CA 53 171 317 31.7  0-1000 41 (77)Colorectal CA 36 3.3 7.7 0.0 0-37.8  5 (14) Breast CA 30 3.7 10.1 0.00-53.5 2 (7) Ovarian CA 15 1.8 4.3 0.0 0-16.5 1 (7) Lymphoma 19 12.344.2 0.0 0-194  1 (5)

An ROC curve (not shown) was constructed with the data from Table 14.Examining a total of 283 patients, including 53 with pancreaticcarcinoma, and comparing the presence of circulating PAM4 antigen inpatients with pancreatic cancer to all other samples, the ROC curveprovided an AUC of 0.88±0.03 (95% ci, 0.84-0.92) with a P value <0.0001,a highly significant difference for discrimination of pancreaticcarcinoma from non-pancreatic carcinoma samples. Comparing pancreatic CAwith other tumors and normal tissue, the PAM4 based serum assay showed asensitivity of 77% and a specificity of 95%.

A comparison was made of PAM4 antigen concentration in serum samplesfrom normal patients, “early” (stage 1) pancreatic carcinoma and allpancreatic carcinoma samples. The specimens included 13 sera fromhealthy volunteers, 12 sera from stage-1, 13 sera from stage-2 and 25sera from stage-3/4 (advanced) pancreatic carcinoma. A cutoff value of8.8 units/ml (red line) was used, as determined by ROC curve statisticalanalysis. The frequency distribution of PAM4 antigen concentration isshown in FIG. 16, which shows that 92% of “early” stage-1 pancreaticcarcinomas were above the cutoff line for diagnosis of pancreaticcancer. An ROC curve for the PAM4 based assay is shown in FIG. 17, whichdemonstrates a sensitivity of 81.6% and specificity of 84.6% for thePAM4 assay in detection of pancreatic cancer.

These results confirm that an enzyme immunoassay based on PAM4 antibodybinding can detect and quantitate PAM4-reactive antigen in the serum ofpancreatic carcinoma patients. The immunoassay demonstrates highspecificity and sensitivity for pancreatic carcinoma. The majority ofpatients with stage 1 disease were detectable using the PAM4immunoassay.

In conclusion, an immunohistology procedure employing PAM4 antibodyidentified approximately 90% of invasive pancreatic carcinoma and itsprecursor lesions, PanIN, IPMN and MCN. A PAM4 based enzyme immunoassayto quantitate PAM4 antigen in human patient sera showed high sensitivityand specificity for detection of early pancreatic carcinoma. Due to thehigh specificity of PAM4 for pancreatic carcinoma, the mucin biomarkercan also serve as a target for in vivo targeting of imaging andtherapeutic agents. ImmunoPET imaging for detection of “early”pancreatic carcinoma is of use for the early diagnosis of pancreaticcancer, when it can be more effectively treated. Use ofradioimmunotherapy with a humanized PAM4 antibody construct, preferablyin combination with a radio sensitizing agent, is of use for thetreatment of pancreatic cancer.

Example 27 Further Studies of In Vitro Detection of PAM4 Antigen inHuman Serum

In certain embodiments, it is preferred to detect the presence of PAM4antigen and/or to diagnose the presence of pancreatic cancer in asubject by in vitro analysis of samples that can be obtained bynon-invasive techniques, such as blood, plasma or serum samples. Such exvivo analysis may be preferred, for example, in screening procedureswhere there is no a priori reason to believe that an individual has apancreatic tumor in a specific location. The objective of the presentstudy was to develop a reliable, accurate, serum-based assay fordetection of pancreatic cancer at the earliest stages of the disease.

SUMMARY

A PAM4-based immunoassay was used to quantitate antigen in the serum ofhealthy volunteers (N=19), patients with known diagnosis of pancreaticadenocarcinoma (N=68), and patients with a primary diagnosis of chronicpancreatitis (N=29). Sensitivity for the detection of pancreaticadenocarcinoma was 82%, with a false-positive rate of 5% for the healthycontrols. Patients with advanced disease had significantly higherantigen levels than those with early-stage disease (P<0.01), with adiagnostic sensitivity of 91%, 86%, and 62% for stage 3/4 advanceddisease, stage-2, and stage-1, respectively. We also evaluated chronicpancreatitis sera, finding 38% positive for antigen. However, thisobservation was discordant with immunohistochemical findings thatsuggest the PAM4-antigen is not produced by inflamed pancreatic tissue.Furthermore, several of the serum-positive pancreatitis patients, forwhom tissue specimens were available for pathological interpretation,had evidence of neoplastic precursor lesions.

These results show that the PAM4-serum assay may be used to detectearly-stage pancreatic adenocarcinoma, and that positive serum levels ofPAM4-antigen are not derived from inflamed pancreatic tissues, butrather may provide evidence of subclinical pancreatic neoplasia.

Materials and Methods

Human Specimens Sera (N=68) were obtained from patients with a confirmeddiagnosis of pancreatic adenocarcinoma being treated at the JohnsHopkins Medical Center, Baltimore, Md., and stored frozen <5 yrs. Eachof these patients underwent surgical resection of the pancreas,providing an opportunity for accurate diagnosis and staging. For stage-1disease, no neoplastic cells were observed outside of the pancreas.However, patients with pancreatic adenocarcinoma are likely to haveundetected micrometastatic disease at presentation, including thosepatients reported with stage-1 disease. For this reason, we evaluatedfollow-up survival data. All patients described as having stage-1disease survived at least 1 year (time to last recorded follow-upvisit), with a median survival time of 2.70 years (25^(th)percentile=1.32 years) in comparison to the latest SEER data(2002-2006), which reports a 1.42-year median survival for patientshaving stage-1 disease treated by surgical resection.

A total of 29 sera from patients with a diagnosis of chronicpancreatitis were obtained from the Johns Hopkins Medical Center andZeptometrix Corp. (Franklin, Mass.). Healthy volunteers (N=19) providedblood for control specimens at the Center for Molecular Medicine andImmunology. All specimens were de-identified, with the only clinicaldata provided to the investigators being the diagnosis, stage ofdisease, follow-up survival time, and size of the primary tumor.

Reagents A human pancreatic mucin preparation was isolated from CaPan1,a human pancreatic cancer grown as xenografts in athymic nude mice.Briefly, 1 g of tissue was homogenized in 10 mL of 0.1 M ammoniumbicarbonate containing 0.5 M sodium chloride. The sample was thencentrifuged to obtain a supernatant that was fractionated on aSEPHAROSE®-4B-CL column with the void volume material chromatographed onhydroxyapatite. The unadsorbed fraction was dialyzed extensively againstdeionized water and then lyophilized. A 1 mg/mL solution was prepared in0.01 M sodium phosphate buffer (pH, 7.2) containing 0.15 M sodiumchloride (phosphate-buffered saline [PBS]), and used as the stocksolution for the immunoassay standards. A polyclonal, anti-mucinantiserum was prepared by immunization of rabbits, as describedpreviously (Gold et al., Cancer Res 43:235-38, 1983). An IgG fractionwas purified and assessed for purity by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) andmolecular-sieve high-performance liquid chromatography. Murine MA5antibody reactive with the MUC-1 protein core was obtained fromImmunomedics, Inc. (Morris Plains, N.J.). A non-binding isotype-matchedcontrol antibody, Ag8, was purified from the P3×63-Ag8 murine myeloma.

Sample Preparation All assays were performed in a blinded fashion. Toprepare the specimens for immunoassay, 300 μL of serum were placed in a2.0 mL microcentrifuge tube and extracted with an equal volume of1-butanol. The tubes were vortexed vigorously for 2 min at which time300 μL of chloroform were added and the tubes again vortexed for 2 min;this latter step was included in the procedure in order to invert theaqueous and organic layers. The tubes were then centrifuged in amicrofuge at a setting of 12,000 rpm for 5 min. The top aqueous layerwas removed to a clean tube and the sample diluted 1:2 in 2.0% (w/v)casein-sodium salt in 0.1 M sodium phosphate buffer, pH 7.2, containing0.15 M sodium chloride (PBS) for immunoassay.

Enzyme immunoassay The immunoassay was performed in a 96-well polyvinylplate that had been coated with 100 μL of humanized-PAM4 IgG at 20 μg/mLin PBS with incubation at 4° C. overnight. The wells were then blockedby addition of 200 μL of a 2.0% (w/v) solution of casein in PBS andincubated for 1.5 h at 37° C. The blocking solution was removed from thewells and the plate washed 5-times with 250 μL of PBS containing 0.1%(v/v) Tween-20. The standards, or unknown specimens, 100 μL intriplicate, were added to the appropriate wells and incubated at 37° C.for 1.5 h. The plate was then washed 5-times with PBS-Tween-20 as above.

The polyclonal, rabbit anti-mucin antibody, diluted to 5 μg/mL in 1.0%(w/v) casein in PBS containing 50 μg/mL non-specific, human IgG, wasadded to each well and incubated for 1 h at 37° C. The polyclonalantibody was then washed from the wells as above, and peroxidase-labeleddonkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove,Pa.), at a 1:2000 dilution in 1.0% (w/v) casein in PBS, also containing50 μg/mL human IgG, was added to the wells and incubated at 37° C. for 1h. After washing the plate as above, 100 μL of a3,3′,5,5′-tetramethylbenzidine substrate solution were added to thewells and incubated at room temperature for 30 min. The reaction wasstopped by the addition of 50 μL 4.0 N sulfuric acid, and the opticaldensity read at a wavelength of 450 nm using a SPECTRA-MAX® 250spectrophotometer (Molecular Devices, Sunnyvale, Calif.). Because of theconsiderable microheterogeneity of the PAM4-mucin, we chose to reportour results in arbitrary units/mL, based on an initial referencestandard purified from xenografted CaPan-1 human pancreatic tumor.

Immunohistochemistry Paraffin-embedded specimens obtained from theCooperative Human Tissue Network were cut to 4 micron sections onsuperfrost plus adhesive slides (Thermo Scientific, Waltham, Mass.).Tissue sections were then heated to 95° C. for 20 min in a pH 9.0 Trisbuffer, Target Retrieval Solution (Dako, Carpinteria, Calif.), allowedto cool to room temperature, and then quenched with 3% H₂O₂ for 15 minat room temperature. Primary antibodies were then used at 10 μg/mL withan ABC VECTASTAIN® kit (Vector Laboratories, Burlingame, Calif.) forlabeling the tissues. The slides were scored independently by twopathologists using a paradigm consistent with that reported for earlierstudies on biomarkers in pancreatic adenocarcinoma (Gold et al., 2007,Clin Cancer Res 13:7380-87): O-negative, <1% of the tissue was labeled;1-a weak, focal labeling of between 1%-25% of the tissue; 2-a strong,focal labeling of between 1%-25% of the tissue; 3-a weak, diffuselabeling >25% of the tissue; 4-a strong, diffuse labeling >25% of thetissue. Only the appropriate tissue components (e.g., adenocarcinomacells, normal ducts, etc.) were considered for assessment.

Statistical Analyses Standard curves were generated from the immunoassaydata, with regression analyses performed to interpolate concentrationsof the unknown samples (Prism 4.0 software, GraphPad, La Jolla, Calif.).Receiver operating characteristic (ROC) curves were generated by use ofthe Med-Calc statistical software package (version 7.5) (Med-Calc,Mariakerke Belgium). Student's t-test was used to compare variables inany two groups. The Cochran-Armitage test was used to detect a trendbetween detection rates and stage of disease.

Results

Accuracy and precision of the immunoassay A set of control standardswith nominal concentrations of 15.60, 6.20, 2.50, and 1.00 units/mL wasevaluated on several nonconsecutive days (N=7) for determination ofaccuracy and precision. Curve fitting for the standards generally gaveresultant goodness of fit values for r²>0.990. Accuracy was calculatedto be within 8% of the nominal value for the first three concentrations,but fell to approximately 22% for the 1.00 units/mL standard. Linearregression of nominal vs. measured units/mL in this series of controlsgave a trend-line with a slope of 0.965 and y intercept of 0.174(r²=0.999), where a slope of 1.00 with a y intercept of 0.00 wouldconstitute 100% accuracy (FIG-. 18). An average absolute differencebetween nominal and recovered mass equal to 0.190±0.173 units/mL for thetwo lowest concentration standards suggested a minimum absolute error ofapproximately 0.2 units/mL for the EIA. Values for the coefficient ofvariation (CV) were 6.40%, 4.85%, 12.0%, and 66.4%, respectively, forthe 4 control standards. Taken together, the data suggest that thePAM4-immunoassay provides levels of accuracy and reproducibility thatare within the guidelines suggested for an immunoassay measurement of ananalyte; accuracy and precision were within 15% for concentrations abovethe cutoff value (2.40 units/mL), and within 20% at the cutoff value. Tofurther test this, we examined 3 sera, two of which were from healthycontrols, on 3 separate days. The two healthy controls gave averageresults of 0.27±0.06 and 0.30±0.27 units/mL, each of which was close tothe minimum absolute error for the EIA with consequent high CV of 21.65%and 88.19%, respectively. The other patient serum gave an average of19.45±2.51 units/mL with a CV of 12.9%.

Quantitation of antigen in patient sera In a preliminary study reportedin Example 26 above, the PAM4 serum-based immunoassay had an apparentsensitivity of 77% and a specificity of 94% for pancreatic carcinoma. Weevaluated a new group of 24 sera from patients diagnosed with pancreaticadenocarcinoma. Only two of the sera had levels of PAM4-reactive antigenconsidered to be positive. Therefore, we considered and evaluatedreasons why the immunoassay had not performed as expected, including thequality of the immunoassay reagents, the possibility that the antigenwas being degraded and/or removed from the serum, its presence in theform of immune complexes, or being bound by a blocking substance. Wediscovered that there is a substance in fresh human serum and/orspecimens stored frozen for short periods of time (<5 yrs) thatapparently binds to the PAM4-reactive epitope and blocks its binding toPAM4 antibody, thus preventing detection by immunoassay. Percentrecovery of antigen from fresh normal human serum (N=2) spiked withPAM4-antigen at concentrations from 5-20 units/mL was on the order of33% or less.

In a series of reports, Slomiany and co-workers disclosed that gastricmucin had covalenty bound and/or associated lipids and fatty-acids(Slomiany et al., 1984, Arch Biochem Biophys 229:560-67; Slomiany etal., 1986, Biochem Biophys Res Commun 141:387-93; Zalesna et al., 1989,Biochem Int 18:775-84), and that these lipids and fatty acids hadspecific effects upon the physicochemical properties of the mucin.Furthermore, it was reported that fatty-acid synthetase levels andactivity are significantly elevated in pancreatic adenocarcinoma, as isalso the case for other forms of cancer and other pathologic conditions(Walter et al., 2009, Cancer Epidemiol Biomarkers Prey 19:2380-85).Because the blocking substance might be lipid in nature, we performedorganic extraction of sera from the group of 24 pancreaticadenocarcinoma patients that had been stored frozen for <5 years. As wasnoted above, without prior extraction, only 2 of the 24 specimens (8.3%)had levels of PAM4-antigen that were considered positive, whereas afterorganic extraction, 22 of the 24 specimens (92%) had positive levels ofthe PAM4-antigen.

We were also able to re-evaluate, from the study reported in Example 26,10 pancreatic adenocarcinoma patient sera that had been stored frozenfor >15 years to confirm the prior results. With or without extraction,all 10 specimens had levels of antigen that were considered to bepositive. Regression analysis to compare paired results from extractedand non-extracted sera gave a trendline with slope of 1.10 (r²=0.94),demonstrating that with or without extraction of these long-term frozensera, the results were similar. It is considered that long-term storageof the specimens resulted in degradation of the inhibiting substance ordecreased binding to and unmasking of the epitope. All further testingof sera was performed with organic extraction of specimens prior toimmunoassay.

Specimens evaluated for PAM4-reactive antigen included 68 patients withconfirmed pancreatic adenocarcinoma divided by stage: 21 from stage-1;14 from stage-2; and 33 from stages-3 and -4 (advanced). In addition, 19sera collected from healthy adult volunteers and 29 patients diagnosedwith chronic pancreatitis were included as control groups. The maximumconcentration shown in the dot-plot (FIG. 19) was 80 units/mL, becausethere were insufficient volumes of sera to perform additional dilutionstudies. Although a cutoff value of 10.2 units/mL was reported inExample 26 above, because of the use of an organic extraction procedure,as well as differences in the EIA protocol (reagent concentrations,inclusion of human IgG in buffers), we chose to treat the current dataset independently of prior results. A positive cutoff value of 2.4units/mL was calculated by ROC curve statistics (FIG. 20) for thecomparison of all pancreatic adenocarcinoma specimens versus healthyadults. The overall sensitivity for detection of pancreaticadenocarcinoma was 82%, with an area under the curve of 0.92±0.03 (95%CI=0.84-0.97). At this level of sensitivity, a false-positive rate of 5%was observed for the healthy control group, the single positive casehaving 3.65 units/mL of circulating antigen, just above the cutoffvalue. Insufficient volumes of sera precluded CA19-9 immunoassays forcomparison to the PAM4-immunoassay results.

As shown in Table 15, sensitivity for detection of early, stage-1pancreatic adenocarcinoma was relatively high, with 13 of 21 (62%)specimens above the cutoff value. As expected, this detection rate waslower than that observed for the stage-2 (86%) and advanced stage-3 and-4 (91%) patient groups. A statistically significant trend (P<0.01) wasnoted for detection rate vs. stage of disease. We considered that thiswas most likely due to tumor size or burden. The average tumor sizes forstage-1, stage-2, and stage-3/4 groups were 2.14±1.02 cm³, 3.36±1.18cm³, and 3.45±1.06 cm³, respectively. While there was no statisticallysignificant difference in tumor size between the stage-2 and -3/4 groups(P >0.41), a statistically significant difference was observed for eachof these two groups when compared to stage-1 tumor size (P<0.004 orbetter). However, it should be noted that individual tumor size did notcorrelate with antigen concentration in the serum (r²=0.0065).

Specimens reported as Stage-1 could be divided into stage-1A (N=13) andstage-1B (N=8) subgroups based on tumor size, with detection rates of54% and 75%, respectively; however, caution is emphasized since thenumber of patients in each subgroup is small. The average tumor size forstage-1A was 1.41±0.58 cm³ (range: 0.4 cm³-2.0 cm³) and for stage-1B was3.15±0.44 cm³ (range: 2.5 cm³-4 cm³); P<0.001 for comparison of the twogroups. While, on the whole, tumor sizes were smaller in stage-1Adisease than in stage-1B, there was no apparent statistical correlationbetween individual tumor size and concentration of the PAM4-antigen inthe blood (r²=0.03). Furthermore, it is important to note that of the 13stage-1A specimens, 4 of the 7 positive cases had PAM4-antigen levelsconsiderably higher than the cutoff value, with a range of 17.65-32.65units/mL.

TABLE 15 PAM4-reactive antigen in the sera of patients Median T-test N(units/mL) True-Positive (P value)^(a) Total PC 68 9.85 81% <0.001Stage-1 21 4.53 62% <0.002 Stage-1A 13 3.96 54% <0.02 Stage-1B 8 6.0575% <0.02 Stage-2 14 10.39 86% <0.005 Stage-3/4 33 13.37 91% <0.001Chronic Pancreatitis 29 1.28 (38% FP) Healthy Volunteers 19 1.18  (5%FP) ^(a)All comparisons are to healthy volunteers

We also evaluated a set of 29 patient sera with the primary diagnosis ofchronic pancreatitis. At the 2.4 units/mL cutoff established by ROCevaluation of normal and pancreatic adenocarcinoma patients, 11pancreatitis patients (38%) were positive. ROC curve analysis ofpancreatitis sera compared directly to the pancreatic adenocarcinomaspecimens gave an area under the curve of 0.77±0.05 (95% CI=0.68-0.85).The median value for the pancreatitis group was 1.28 units/mL,comparable to the healthy volunteer group (1.18 units/mL), butconsiderably lower (3.5-fold) than the stage-1 pancreatic adenocarcinomagroup (4.53 units/mL). It should be noted that our earlier results forpancreatitis specimens suggested a considerably lower false-positiverate, only 5%. However, those pancreatitis specimens were stored frozenfor less than 5 years, and were not organic phase extracted prior toanalysis.

Biopsy and/or surgical specimens were available from 14 of the chronicpancreatitis specimens, 6 of which were from patients who wereconsidered positive for circulating PAM4 antigen. In 3 of these 6positive cases, precursor lesions were identified within the tissuesections. It was then considered whether the positive serum test was dueto pancreatitis or the presence of neoplastic precursor lesions. Weperformed immunohistochemistry on an additional 30 biopsy specimens frompatients diagnosed with pancreatitis. Of the 30 specimens, one frankinvasive pancreatic adenocarcinoma and one large PanIN-2-3 lesion wereidentified (in separate specimens) by use of PAM4 staining, whilesurrounding acinar-ductal metaplasia (ADM) and normal tissues werenegative (data not shown). Of the remaining 28 specimens, 19 hadsufficient parenchyma to be evaluated, 16 of which had evidence of ADM.PAM4 was negative in all but two of these cases, and in each of thesegave only a very focal, weak labeling of ADM within the specimens (datanot shown).

Discussion

Studies reported in Example 26 that employed both immunohistology oftissue specimens and EIA of circulating antigen demonstrated that thePAM4-reactive epitope is a biomarker for invasive pancreaticadenocarcinoma and is expressed at the earliest stages of pancreaticneoplasia (i.e., PanIN-1). It was not detectable within normalpancreatic tissues (ducts, acinar and islet cells), nor the majority ofnon-pancreatic cancers examined (breast, lung, gastric, and others).Thus, an elevation of the PAM4-epitope concentration in the serumprovided a high positive likelihood ratio of 16.8 for pancreaticadenocarcinoma. Missing from the prior study was clinical informationregarding the stage of disease. Consequently, we could not evaluate thevalue of the immunoassay for detection of potentially curable earlydisease until now.

We report herein that PAM4-based EIA using serum samples can detectpatients having early-stage pancreatic adenocarcinoma, and can provideaccurate discrimination from disease-free individuals. The assay'ssensitivity for detection of early pancreatic adenocarcinoma was 62% forpatients with stage-1 and 86% for patients with stage-2 disease andserum levels generally increased with advancing stage of disease. A highpercentage of patients with stage-1 and -2 disease are clinicallyasymptomatic. We conclude that detection of tumor growth at these earlystages using a PAM4 serum assay could provide improved prospects forsurvival.

The cancer patients in this study all underwent surgical resection,providing an opportunity to accurately stage each patient. However, manypatients with pancreatic cancer are suspected of having micrometastaticdisease at presentation, even if they do not havehistologically-apparent regional lymph node involvement. This highlightsa general problem in the study of early detection, particularly with alow-incidence disease such as pancreatic adenocarcinoma. The accrual ofspecimens that are well-defined is problematic. Further complicating theissue is that many of these pancreatic cancers occur in the presence ofchronic pancreatitis, cholecystitis, and neoplastic precursor lesions,amongst other conditions.

Of 29 sera with a primary diagnosis of chronic pancreatitis, 38% wereidentified as positive for PAM4-antigen. However, several of theseserum-positive patients, for whom tissue specimens for pathologicalinterpretation were available, had evidence of neoplastic precursorlesions. Furthermore, a discrepancy was observed in the comparison oftissue reactivity by immunohistology and serum levels of antigen byimmunoassay. By immunohistochemistry, only 10% of the evaluablespecimens showed evidence of PAM4 staining within the ADM, although thiswas at considerably lower intensity than observed for the overwhelmingmajority of pancreatic adenocarcinoma specimens (Gold et al., 2007, ClinCancer Res 13:7380-87). Therefore, the results suggest that positivelevels of PAM4-antigen within the serum may not be derived from inflamedpancreatic tissues, but rather could provide evidence of subclinicalpancreatic neoplasia, such as PanIN lesions, and that, at the veryleast, positive results provide the rationale for clinical follow-up ofthese patients.

Findings from genetically-engineered animal models of pancreaticadenocarcinoma suggest that human pancreatic neoplasia may arise beforethe PanIN-1 lesion (Leach, 2004, Cancer Cell 5:7-11). ADM was theearliest change observed in the mutant KRAS targeted model described byZhu et al. (2007, Am J Pathol 171:263-73). On the other hand, Shi et al.(2009, Mol Cancer Res 7:230-36) reported that although KRAS genemutations can occur within ADM, they occur predominantly within ADM thatare associated with PanIN lesions. The authors suggest this may occur byretrograde extension of the PanIN to the surrounding ADM. As yet, thereis no conclusive evidence that ADM progress to PanIN. The fact that PAM4is reactive with ADM in two patients with pancreatitis is of interest.

At the present time, screening the general population for pancreaticcancer is not considered medically or economically worthwhile, becausethe disease is simply too infrequent. However, there is considerableinterest in screening patients predicted to have an increased risk ofdeveloping pancreatic adenocarcinoma. Several studies have demonstratedthat screening individuals with strong family histories of pancreaticcancer can identify precursor neoplasms of the pancreas that areamenable to surgical resection (Canto et al., 2006, Clin GastroenterolHepatol 4:766-81; Canto, 2005, Clin Gastroenterol Hepatol 3:S46-58;Brentnall et al., 1999, Ann Intern Med 131:247-55). For example,relatives of pancreatic cancer patients have a significantly higher riskof developing pancreatic cancer than the general population (Shi et al.,2009, Arch Pathol Lab Med 133:365-74). A small percentage of patientswith familial pancreatic cancer harbor mutations of PALB2 (partner andlocalizer of BRCA2), a susceptibility gene for pancreatic cancer(Tischkowitz et al., 2009, Gastroenterology 137:1183-86). Similarly,patients with long-standing chronic pancreatitis are at increased riskof developing pancreatic cancer, and the risk is over 30%, amongpatients with early-onset (teenage) hereditary pancreatitis (Lowenfelset al., 1993, New Eng J Med 328:1433-37; Lowenfels et al., 1997, J NatlCancer Inst 89:442-46). A 20- to 34-fold higher risk has been observedin individuals with familial atypical multiple mole (FAMMM) syndrome(Rutter et al., 2004, Cancer 101:2809-16). Also, several studies haveshown a significantly increased risk of developing pancreatic cancer indiabetic individuals who meet certain criteria (Pannala et al., 2009,Lancet Oncol 10:88-95). Longitudinal surveillance of these patients byuse of the PAM4-immunoassay may provide for early detection ofneoplasia. A second potential use of the immunoassay could be as a meansto detect recurrence of disease post-therapy, and in particular,following surgical resection for those patients where the tumor issupposedly confined to the pancreas.

The relatively high specificity of the PAM4 antibody provides a means totarget both imaging and therapeutic agents with high tumor uptake andhigh tumor/nontumor ratios. We have demonstrated PAM4's potential asboth a directly-radiolabeled or bispecific, pretargeting reagent fornuclear imaging and radioimmunotherapy of pancreatic cancer. Also,initial results of a clinical phase 1b trial to evaluate a fractionateddosing of ⁹⁰Y-PAM4 whole IgG (clivatuzumab tetraxetan), in combinationwith a radiosensitizing regimen of gemcitabine, were reported recently(Pennington et al., 2009, J Clin Oncol 27:15s, abstract 4620). Of 22patients with stage-3/4 disease (mostly stage-4), 68% showed evidence ofdisease control, with 23% of patients having partial responses based onRECIST criteria. Thus, positive results by the PAM4-based immunoassayprovides a rationale to pursue PAM4-targeted imaging and therapy, thusproviding a personalized therapy.

The PAM4-based immunoassay can identify the majority of pancreaticadenocarcinoma patients of all stages. Although a direct comparison withCA19.9 was not possible in the current study, a prior comparison of thetwo biomarkers in a limited set of pancreatic adenocarcinoma sera (N=41)demonstrated a statistically significant difference (P<0.01) withPAM4-antigen levels positive in 71% of patient specimens andCA19.9-antigen levels positive in 59% of specimens. In general, it isthought that CA19.9 lacks the sensitivity and specificity to provide forearly detection and/or diagnosis of pancreatic adenocarcinoma. However,the assay does have its use for management with continued elevation inCA19.9 serum levels post treatment indicative of a poor prognosis.Similarly, we recently reported in abstract form (Pennington et al.,2009, J Clin Oncol 27:15s, abstract 4620), the use of circulatingPAM4-antigen levels for prediction of anti-tumor response.

These results show that the conditions under which specimens are stored(e.g., the length of time they are kept frozen) can have significanteffects upon accessibility of the epitope under study. For thePAM4-based immunoassay, a fatty acid or lipid substance may be able tobind the specific epitope and interfere with the immunoassay. However,it is also possible this material was a low-molecular weight peptide orother substance soluble in organic solvents. The ability to remove thissubstance by organic extraction of the serum makes the PAM4-immunoassayreproducible. In addition, the question is raised as to the biologicalsignificance of the circulating inhibitor:PAM4 antigen interaction.However, when using the PAM4 antibody as an in vivo targeting agent(e.g., radioimmunotherapy), the presence of circulating PAM4-antigen isnot a factor, since targeting of radiolabeled-PAM4 to sites of tumorgrowth has been observed in the majority of patients evaluated to date.Thus, it appears that the PAM4-antigen within tumor is free of theblocking substance.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the products, compositions,methods and processes of this invention. Thus, it is intended that thepresent invention cover such modifications and variations, provided theycome within the scope of the appended claims and their equivalents.

1. A method of detecting or diagnosing pancreatic cancer comprising: a)obtaining a blood, serum or plasma sample from an individual; and b)performing an immunoassay with an anti-pancreatic cancer antibody todetect the presence of a pancreatic cancer mucin in the sample; whereinthe presence of the pancreatic cancer mucin is indicative of pancreaticcancer in the individual and the serum immunoassay can detect earlystage pancreatic cancer.
 2. The method of claim 1, wherein the serumimmunoassay can detect stage 1A, stage 1B and stage 2 pancreatic cancer.3. The method of claim 1, wherein the serum immunoassay can detect 80%or more of total pancreatic cancers.
 4. The method of claim 1, whereinthe serum immunoassay can detect 60% or more of stage 1 pancreaticcancers.
 5. The method of claim 1, wherein the serum immunoassay candetect 54% of stage 1A pancreatic cancers.
 6. The method of claim 1,wherein the serum immunoassay can detect 75% of stage 1B pancreaticcancers.
 7. The method of claim 1, wherein the serum immunoassay candetect 86% of stage 2 pancreatic cancers.
 8. The method of claim 1,wherein the serum immunoassay can detect 90% or more of stage 3 andstage 4 pancreatic cancers.
 9. The method of claim 1, wherein the falsepositive rate of the serum immunoassay for control individuals withoutpancreatic cancer or pancreatitis is 5% or less.
 10. The method of claim1, wherein the false positive rate of the serum immunoassay forindividuals with chronic pancreatitis is 38% or less.
 11. The method ofclaim 1, wherein the serum immunoassay can detect pancreaticadenocarcinoma in asymptomatic individuals.
 12. The method of claim 1,wherein the sample is a serum sample and the method further comprisesperforming an organic phase extraction on the serum sample before theimmunoassay is performed.
 13. The method of claim 12, wherein theorganic phase is butanol.
 14. The method of claim 1, wherein theimmunoassay detects the presence of PanIN-1A, PanIN-1B, PanIN-2,invasive pancreatic adenocarcinoma, pancreatic carcinoma, mucinous cystneoplasms (MCN), intrapancreatic mucinous neoplasms (IPMN) andintraductal papillary mucinous neoplasia.
 15. A method of detecting ordiagnosing pancreatic cancer comprising: a) administering to anindividual an antibody construct comprising at least one anti-pancreaticcancer antibody or antigen-binding fragment that binds to the sameepitope as or competes for binding to pancreatic cancer mucin with achimeric PAM4 (cPAM4) antibody that comprises the light chain variableregion CDR sequences CDR1 (SASSSVSSSYLY, (SEQ ID NO:1); CDR2 (STSNLAS,SEQ ID NO:2); and CDR3 (HQWNRYPYT, SEQ ID NO:3); and the heavy chainvariable region CDR sequences CDR1 (SYVLH, SEQ ID NO:4); CDR2(YINPYNDGTQYNEKFKG, SEQ ID NO:5) and CDR3 (GFGGSYGFAY, SEQ ID NO:6); andb) detecting the antibody construct bound to pancreatic cancer cells.16. The method of claim 15, wherein the antibody construct is exposed tothe sample in vivo or in vitro.
 17. The method of claim 15, wherein theanti-pancreatic cancer antibody or fragment thereof binds to a linearpeptide comprising the amino acid sequence WTWNITKAYPLP (SEQ ID NO: 7)or to a cyclic peptide comprising the amino acid sequence ACPEWWGTTC(SEQ ID NO 8).
 18. The method of claim 15, wherein the antibodyconstruct is a bispecific or multispecific antibody comprising (i) atleast one PAM4 antibody or antigen-binding fragment thereof; and (ii) atleast one antibody or fragment that binds to a hapten on a targetableconstruct; and the method further comprises administering a targetableconstruct attached to at least one diagnostic agent
 19. The method ofclaim 15, wherein the anti-pancreatic cancer antibody or antigen-bindingfragment thereof is conjugated to at least one diagnostic agent.
 20. Themethod of claim 19, wherein the diagnostic agent is selected from thegroup consisting of a radionuclide, a contrast agent, a fluorescentagent, a chemiluminescent agent, a bioluminescent agent, a paramagneticion, an enzyme and a photoactive diagnostic agent.
 21. The method ofclaim 20, wherein the diagnostic agent is a radionuclide selected fromthe group consisting of ¹¹⁰In, ¹¹¹In, ¹⁷⁷Lu, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu,⁶⁷Cu, ⁶⁷Ga, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr, ^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹²⁰I, ¹²³I, ¹²⁴I,¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ³²P, ¹¹C, ¹³N, ¹⁵O, ¹⁸⁶Re, ¹⁸⁸Re, ⁵¹M, ^(52m)Mn,⁵⁵Co, ⁷²As, ⁷⁵Br, ⁷⁶Br, ^(82m)Rb, ⁸³Sr, or other gamma-, beta-, orpositron-emitters.
 22. The method of claim 21, wherein the radionuclideis ¹⁸F and the method further comprising PET imaging.
 23. The method ofclaim 20, wherein the paramagnetic ion is selected from the groupconsisting of chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and erbium (III).
 24. The method ofclaim 20, wherein the diagnostic agent is a fluorescent labelingcompound selected from the group consisting of fluoresceinisothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin,o-phthaldehyde and fluorescamine, or a chemiluminescent labelingcompound selected from the group consisting of luminol, isoluminol, anaromatic acridinium ester, an imidazole, an acridinium salt and anoxalate ester, or a bioluminescent compound selected from the groupconsisting of luciferin, luciferase and aequorin.
 25. The method ofclaim 15, wherein the method is used in intraoperative, endoscopic, orintravascular procedure.
 26. The method of claim 15, wherein theantibody construct is a DNL (dock and lock) construct comprising (i) atleast one copy of a chimeric, human or humanized PAM4 antibody orfragment thereof, wherein the PAM4 antibody or fragment thereofcomprises the light chain variable region CDR sequences CDR1(SASSSVSSSYLY, (SEQ ID NO:1); CDR2 (STSNLAS, SEQ ID NO:2); and CDR3(HQWNRYPYT, SEQ ID NO:3); and the heavy chain variable region CDRsequences CDR1 (SYVLH, SEQ ID NO:4); CDR2 (YINPYNDGTQYNEKFKG, SEQ IDNO:5) and CDR3 (GFGGSYGFAY, SEQ ID NO:6); and (ii) at least one copy ofa hapten-binding antibody or fragment thereof.
 27. The method of claim26, wherein the hapten-binding antibody is an h679 antibody or an h734antibody.
 28. The method of claim 26, wherein the DNL constructcomprises two copies of a PAM4 Fab fragment, each attached to a DDDmoiety, and one copy of an h679 Fab fragment attached to an AD moiety,wherein the two copes of the DDD moiety form a dimer that binds to theAD moiety to form the DNL construct.